Efficient method for producing compositions enriched in total phenols

ABSTRACT

This invention provides a process for the preparation of compositions enriched in total phenols from a crude plant extract. The process includes a novel column purification step using a brominated polystyrene resin. This invention also includes compositions enriched in total phenols. The enriched compositions are characterized as containing monomeric, oligomeric and polymeric phenols and having HPLC chromatograms substantially as set forth in FIGS.  10 - 13 . This invention encompasses methods of using the total phenol-enriched compositions for treating warm-blooded animals, including humans, infected with paramyxovaridae such as respiratory syncytial virus, orthomyoxovaridae such as influenza A, B, and C, parainfluenza, Herpes viruses such as HSV-1 and HSV-2, and Flaviviruses such as West Nile Virus, and for treating inflammation such as caused by arthritis, stress and digestive disease.

RELATED APPLICATIONS

[0001] This application is a Continuation-in-Part application of U.S.patent application Ser. No. 09/943,158, filed Aug. 30, 2001, andentitled “Efficient Method for Producing Compositions Enriched inAnthocyanins,” which claims priority to U.S. Provisional Application No.60/229,205, filed Aug. 31, 2000, and entitled “Efficient Method forProducing Compositions Enriched in Anthocyanins.”

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] The present invention relates to the extraction and purificationof flavonoid compounds from plant material, and more specifically to theproduction of compositions enriched in total phenols.

[0004] 2. Description of the Prior Art

[0005] Flavonoid compounds are present in all aerial parts of plants,with high concentrations found in the skin, bark, and seeds. Suchcompounds are also found in numerous beverages of botanical origin, suchas tea, cocoa, and wine. The flavonoids are a member of a larger familyof compounds called polyphenols. That is, these compounds contain morethan one hydroxyl group (OH) on one or more aromatic rings. The physicaland chemical properties, analysis, and biological activities ofpolyphenols and particularly flavonoids have been studied for manyyears.

[0006] Anthocyanins are a particular class of naturally occurringflavonoid compounds that are responsible for the red, purple, and bluecolors of many fruits, vegetables, cereal grains, and flowers. Forexample, the colors of fruits such as blueberries, bilberries,strawberries, raspberries, boysenberries, marionberries, cranberries,elderberries, etc. are due to many different anthocyanins. Over 300structurally distinct anthocyanins have been identified in nature.Because anthocyanins are naturally occurring, they have attracted muchinterest for use as colorants for foods and beverages.

[0007] Recently, the interest in anthocyanin pigments has intensifiedbecause of their possible health benefits as dietary antioxidants. Forexample, anthocyanin pigments of bilberries (Vaccinium myrtillus) havelong been used for improving visual acuity and treating circulatorydisorders. There is experimental evidence that certain anthocyanins andother flavonoids have anti-inflammatory properties. In addition, thereare reports that orally administered anthocyanins are beneficial fortreating diabetes and ulcers and may have antiviral and antimicrobialactivities. The chemical basis for these desirable properties offlavonoids is believed to be related to their antioxidant capacity.Thus, the antioxidant characteristics associated with berries and otherfruits and vegetables have been attributed to their anthocyanin content.

[0008] Proanthocyanidins, also known as “oligomeric proanthocyanidins,”“OPCs,” or “procyanidins,” are another class of naturally occurringflavonoid compounds widely available in fruits, vegetables, nuts, seeds,flowers, and barks. Proanthocyanidins belong to the category known ascondensed tannins. They are the most common type of tannins found infruits and vegetables, and are present in large quantities in the seedsand skins. In nature, mixtures of different proanthocyanidins arecommonly found together, ranging from individual units to complexmolecules (oligomers or polymers) of many linked units. The generalchemical structure of a polymeric proanthocyanidin comprises linearchains of flavonoid 3-ol units linked together through common C(4)-C(6)and/or C(4)-C(8) bonds. ¹³C NMR has been useful in identifying thestructures of polymeric proanthocyanidins, and recent work haselucidated the chemistry of di-, tri-, and tetrameric proanthocyanidins.Larger oligomers of the flavonoid 3-ol units are predominant in mostplants and are found with average molecular weights above 2,000 Daltonsand containing 6 or more monomer units (Newman, et al., Mag. Res. Chem.,25:118 (1987)).

[0009] Considerable recent research has explored the therapeuticapplications of proanthocyanidins, which are primarily known for theirantioxidant activity. However, these compounds have also been reportedto demonstrate antibacterial, antiviral, anticarcinogenic,anti-inflammatory, anti-allergic, and vasodilatory actions. In addition,they have been found to inhibit lipid peroxidation, plateletaggregation, capillary permeability and fragility, and to affect enzymesystems including phospholipase A2, cyclooxygenase, and lipoxygenase.For example, proanthocyanidin monomers (i.e., anthocyanins) and dimershave been used in the treatment of diseases associated with increasedcapillary fragility and have also been shown to have anti-inflammatoryeffects in animals (Beladi, et al., Ann. N.Y. Acad. Sci., 284:358(1977)). Based on these reported findings, oligomeric proanthocyanidins(OPCs) may be useful components in the treatment of a number ofconditions (Altern. Med. Rev. 5(2):144-151 (2000)).

[0010] Proanthocyanidins may also protect against viruses. In in vitrostudies, proanthocyanidins from witch hazel (Hamamelis virginiana)killed the Herpes simplex 1 (HSV-1) virus (Erdelmeier, C. A., Cinatl,J., Plant Med. June: 62(3):241-5 (1996); DeBruyne, T., Pieters, L., J.Nat. Prod. July: 62(7):954-8 (1999)). Another study was carried out todetermine the structure-activity relationships of the antiviral activityof various tannins. It was found that the more condensed the chemicalstructure, the greater the antiviral effect (Takechi, M., et al.,Phytochemistry, 24:2245-50 (1985)). In another study, proanthocyanidinswere shown to have anti-Herpes simplex activity in which the 50 percenteffective doses needed to reduce herpes simplex plaque formation weretwo to three orders of magnitude less than the 50 percent cytotoxicdoses (Fukuchi, K., et al., Antiviral Res., 11:285-298 (1989)).

[0011] Cyclooxygenase (COX-1, COX-2) or prostaglandin endoperoxide Hsynthase (PGHS-1, PGHS-2) enzymes are widely used to measure theanti-inflammatory effects of plant products (Bayer, T., et al.,Phytochemistry, 28:2373-2378 (1989); and Goda, Y., et al., Chem. Pharm.Bull., 40:2452-2457 (1992)). COX enzymes are the pharmacological targetsites for nonsteroidal anti-inflammatory drugs (Humes, J. L., et al.,Proc. Natl. Acad. Sci. U.S.A., 78:2053-2056 (1981); and Rome, L. H., etal., Proc. Natl. Acad. Sci. U.S.A., 72:4863-4865 (1975)). Two isozymesof cyclooxygenase involved in prostaglandin synthesis arecyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) (Hemler, M., etal., J. Biol. Chem., 25:251, 5575-5579 (1976)). It is hypothesized thatselective COX-2 inhibitors are mainly responsible for anti-inflammatoryactivity (Masferrer, J. L., et al., Proc. Natl. Acad. Sci. U.S.A.,91:3228-3232 (1994)). Flavonoids are now being investigated asanti-inflammatory substances, as well as for their structural featuresfor cyclooxygenase (COX) inhibition activity.

[0012] Due to the above characteristics and benefits of anthocyanins andproanthocyanidins, much effort has been put forth toward extractingthese compounds from fruits, vegetables, and other plant sources. Inaddition to proanthocyanidins and anthocyanins, plants, fruits, andvegetables also contain other compounds such as mineral salts, commonorganic acids such as citric or tartaric acid, carbohydrates, flavonoidglycosides and catechins. It is often desirable to separate theanthocyanins and proanthocyanidins from other naturally occurringcompounds. Anthocyanins have been extracted from plants and fruits byvarious procedures. One method of extracting anthocyanins employs theaddition of bisulfate to form zwitterionic species. The extract ispassed through an ion exchange column which adsorbs the zwitterionicanthocyanin adducts, and the adsorbed anthocyanins are eluted from theresin with acetone, alkali, or dimethylformamide (DMF). Disadvantages ofthis process include the presence of bisulfate, which interferes withadsorption of anthocyanins, thereby requiring multiple columnadsorptions. Elution with alkali degrades the anthocyanins considerably,while DMF is not a recognized food additive and therefore must becompletely removed before the anthocyanins can be added to any foodproducts.

[0013] In order to capture these flavonoid compounds, well-defined andprecise processing and separation techniques are needed.Nafisi-Movaghar, et al. in U.S. Pat. No. 5,912,363 describe a method forthe extraction and purification of proanthocyanidins from plant materialcomprising heating an aqueous mixture of plant material, filtering theaqueous solution through an ultrafiltration membrane to remove largermolecular weight polymers and particulates to produce a permeatecontaining extracted proanthocyanidins, separating the proanthocyanidinsfrom the liquid by contacting the permeate with an adsorbent materialwhich is capable of releasably retaining the proanthocyanidins, andeluting the retained proanthocyanidins with a polar solvent. However,this method uses a very hot extraction temperature, which can causedegradation of the proanthocyanidins. Further, the ultrafiltrationremoves some of the low molecular weight polyphenolic material from thefinal product.

[0014] Many processes known in the art for extracting and isolatingproanthocyanidins and/or anthocyanins from various plant materials usetoxic and/or environmentally hazardous materials. Consequently, thecurrent methods available for isolating and purifying proanthocyanidinsare not easily scaled up to an efficient commercial process wheredisposal considerations of various chemicals and solvents play animportant role in the overall feasibility of the process. Further,proanthocyanidins and anthocyanins must be isolated in a manner thatminimizes their natural tendency toward degradation.

[0015] There is still a need, therefore, for an efficient process forisolating and purifying compositions containing phenolic compounds suchas proanthocyanidins for uses in nutraceuticals and pharmaceuticals thatis cost-effective, scalable, economically sound, does not require theuse of toxic solvents or reagents, and isolates the phenolic compoundsfrom plant material in a manner that minimizes their tendency towarddegradation.

SUMMARY OF THE INVENTION

[0016] The present invention provides simplified and economic methodsfor the extraction, isolation, and purification of compositions enrichedin total phenols. More specifically, one aspect of this inventionprovides a method of preparing compositions enriched in total phenolscomprising: (a) providing a crude extract of one or more plant materialsthat contain phenolic compounds, said extract comprisingproanthocyanidins, anthocyanins, other small phenolics and non-phenoliccompounds; (b) filtering the crude extract; (c) contacting the crudeextract with a brominated polystyrene resin which releasably adsorbssaid phenols but does not substantially retain the non-phenoliccompounds; (d) washing said resin with a wash eluent to elute saidnon-phenolic compounds; (e) eluting the resin with a first eluent andcollecting a first fraction containing phenols; (f) eluting the resinwith a second eluent and collecting a second fraction containingphenols; and (g) isolating the fractions from step (e) or from step (f)or combining said fractions from steps (e) and (f) to obtain acomposition enriched in total phenols and substantially depleted of saidnon-phenolic compounds. This invention further provides totalphenol-enriched compositions isolated by the methods of this invention.

[0017] This invention further provides methods of fractionating thetotal phenol-enriched compositions to separate polar proanthocyanidinsfrom non-polar proanthocyanidins. This invention further providescompositions enriched in polar proanthocyanidins and compositionsenriched in non-polar proanthocyanidins. The polar proanthocyanidinswere found to have biological activities that are different than thenon-polar proanthocyanidins.

[0018] When the total phenol-enriched compositions of this invention areanalyzed by reversed-phase HPLC on a C-18 lipophilic column,characteristic sets of elution peaks of compounds absorbing at 280 nmand 510 nm are observed. More specifically, the total phenol-enrichedcompositions of this invention are characterized as having acharacteristic set of elution peaks in the region between 60 and 75minutes in an HPLC trace substantially as illustrated in FIGS. 10-13when the HPLC analysis is performed as described herein.

[0019] When the total phenol-enriched compositions of this invention areanalyzed by IR spectrometry, characteristic absorption peaks ofcompounds substantially as shown in FIGS. 33-40 are observed. Thecompositions of this invention are useful as nutraceuticals andpharmaceuticals. For example, the compositions of this invention areuseful as anti-infective (e.g., antiviral, anti-UTI and antimicrobial)agents and as anti-inflammatory agents.

[0020] The foregoing and other features, utilities and advantages of theinvention will be apparent from the following more particulardescriptions of preferred embodiments of the invention and asillustrated in the accompanying drawings and as particularly pointed outin the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The accompanying drawings, which are incorporated herein and forma part of the specification, illustrate non-limiting embodiments of thepresent invention, and together with the description, serve to explainthe principles of the invention.

[0022] In the Drawings:

[0023]FIG. 1 is a flow chart of a method for preparing a totalphenol-enriched composition according to the method of this invention.

[0024]FIG. 2 is an HPLC chromatogram at 510 nm of a totalphenol-enriched composition (“fraction 3”) prepared from bilberries.

[0025]FIG. 3 is an HPLC chromatogram at 280 nm of a totalphenol-enriched composition (“fraction 3”) prepared from bilberries.

[0026]FIG. 4 is an HPLC chromatogram at 510 nm of a totalphenol-enriched composition (“fraction 3”) prepared from blueberries.

[0027]FIG. 5 is an HPLC chromatogram at 280 nm of a totalphenol-enriched composition (“fraction 3”) prepared from blueberries.

[0028]FIG. 6 is an HPLC chromatogram at 280 nm of a filtered elderberryextract.

[0029]FIG. 7 is an HPLC chromatogram at 510 nm of a filtered elderberryextract.

[0030]FIG. 8 is an HPLC chromatogram at 280 nm of a first fractioneluted during column loading of a filtered elderberry extract.

[0031]FIG. 9 is an HPLC chromatogram at 510 nm of a first fractioneluted during column loading of a filtered elderberry extract.

[0032]FIG. 10 is an HPLC chromatogram at 280 nm of a third fractioneluted with 70% ethanol during column purification of an elderberryextract on a brominated polystyrene resin.

[0033]FIG. 11 is an HPLC chromatogram at 510 nm of a third fractioneluted with 70% ethanol during column purification of an elderberryextract on a brominated polystyrene resin.

[0034]FIG. 12 is an HPLC chromatogram at 280 nm of a fourth fractioneluted with 90% ethanol during column purification of an elderberryextract on a brominated polystyrene resin.

[0035]FIG. 13 is an HPLC chromatogram at 510 nm of a fourth fractioneluted with 90% ethanol during column purification of an elderberryextract on a brominated polystyrene resin.

[0036]FIG. 14 is an HPLC chromatogram using an alternate HPLC method ofthe proanthocyanidins standard prepared as described in Example 10.

[0037]FIG. 15 is a flow chart of a method for separating polarproanthocyanidins from non-polar proanthocyanidins.

[0038]FIG. 16 is an HPLC chromatogram at 280 nm of a filtered elderberryextract.

[0039]FIG. 17 is an HPLC chromatogram at 280 nm of an elderberry polarproanthocyanidin composition (“fraction 5”) isolated from the combinedflow-through and wash fractions from a VLC C-18 column.

[0040]FIG. 18 is an HPLC chromatogram at 280 nm of an elderberrynon-polar proanthocyanidin composition (“fraction 6”) isolated in the60% methanol eluent from a VLC C-18 column.

[0041]FIG. 19 is an HPLC chromatogram at 280 nm of an elderberry polarproanthocyanidin composition (“fraction 7”) isolated aftersemi-preparative HPLC purification.

[0042]FIG. 20 is a ¹³C NMR spectrum of an elderberry polarproanthocyanidin composition (“fraction 7”) after purification bysemi-preparative HPLC.

[0043]FIG. 21 is an HPLC chromatogram at 280 nm of an elderberrynon-polar proanthocyanidin composition (“fraction 6”) isolated duringVLC chromatography on C-18 media and before purification on a SephadexLH-20 column, in which the non-proanthocyanidin peaks are marked with anasterisk.

[0044]FIG. 22 is an HPLC chromatogram at 280 nm of the elderberrynon-polar proanthocyanidin composition (“fraction 8”) after purificationon a Sephadex LH-20 column.

[0045]FIG. 23 is an HPLC chromatogram at 368 nm of an elderberrynon-polar proanthocyanidin composition (“fraction 6”) isolated duringVLC chromatography on C-18 media and before purification on a SephadexLH-20 column.

[0046]FIG. 24 is an HPLC chromatogram at 368 nm of an elderberrynon-polar proanthocyanidin composition (“fraction 8”) after purificationon a Sephadex LH-20 column.

[0047]FIG. 25 is a ¹³C NMR spectrum of an elderberry non-polarproanthocyanidin composition (“fraction 8”) after purification on aSephadex LH-20 column.

[0048]FIG. 26 is an HPLC chromatogram at 280 nm of a blueberry polarproanthocyanidin composition (“fraction 5”) isolated during VLCchromatography on C-18 media and before semi-preparative HPLCpurification.

[0049]FIG. 27 is an HPLC chromatogram at 280 nm of a blueberry polarproanthocyanidin composition (“fraction 7”) after purification bysemi-preparative HPLC.

[0050]FIG. 28 is an HPLC chromatogram at 280 nm of a blueberry non-polarproanthocyanidin composition (“fraction 6”) isolated during VLCchromatography on C-18 media and before semi-preparative HPLCpurification.

[0051]FIG. 29 is an HPLC chromatogram at 280 nm of a blueberry non-polarproanthocyanidin composition (“fraction 8”) after purification bysemi-preparative HPLC.

[0052]FIG. 30 is an HPLC chromatogram at 280 nm of a plum polarproanthocyanidin composition (“fraction 5”) isolated during VLCchromatography on C-18 media and before semi-preparative HPLCpurification.

[0053]FIG. 31 is an HPLC chromatogram at 280 nm of a plum polarproanthocyanidin composition (“fraction 7”) after purification bysemi-preparative HPLC.

[0054]FIG. 32 is an HPLC chromatogram at 280 nm of a plum non-polarproanthocyanidin composition (“fraction 6”) isolated during the 40% and70% methanol elution from a VLC C-18 column.

[0055]FIG. 33 is an IR spectrum of a purified elderberry polarproanthocyanidin composition (“fraction 7”).

[0056]FIG. 34 is an IR spectrum of a purified elderberry non-polarproanthocyanidin composition (“fraction 8”).

[0057]FIG. 35 is an IR spectrum of a purified cranberry non-polarproanthocyanidin composition (“fraction 8”).

[0058]FIG. 36 is an IR spectrum of a purified cranberry polarproanthocyanidin composition (“fraction 7”).

[0059]FIG. 37 is an IR spectrum of a purified blueberry polarproanthocyanidin composition (“fraction 7”).

[0060]FIG. 38 is an IR spectrum of a purified blueberry non-polarproanthocyanidin composition (“fraction 8”).

[0061]FIG. 39 is an IR spectrum of a purified plum polarproanthocyanidin composition (“fraction 7”).

[0062]FIG. 40 is an IR spectrum of a purified plum non-polarproanthocyanidin composition (“fraction 6”).

[0063]FIG. 41 is an HPLC chromatogram at 280 nm of a cranberry polarproanthocyanidin composition (“fraction 5”) before semi-preparative HPLCpurification.

[0064]FIG. 42 is an HPLC chromatogram at 280 nm of a cranberry polarproanthocyanidin composition (“fraction 7”) after semi-preparative HPLCpurification.

[0065]FIG. 43 is an HPLC chromatogram at 280 nm of a cranberry non-polarproanthocyanidin composition (“fraction 6”).

DETAILED DESCRIPTION OF THE INVENTION

[0066] This invention provides methods for preparing compositionsenriched in total phenols from plant materials that naturally containphenolic compounds such as anthocyanins and proanthocyanidins. Themethod of this invention further provides compositions enriched in totalphenols.

[0067] As used herein, the term “extract” refers to a substance derivedfrom a plant source that naturally contains phenolic compounds,including extracts prepared from the whole plant or from various partsof the plant, such as the fruits, leaves, stems, roots, bark, etc. Thus,the method of this invention is not limited to the particular part ofthe plant used to prepare the extract. The present method can use anysource of anthocyanins and proanthocyanidins, most typically frombotanically derived plant material such as seeds, fruits, skins,vegetables, nuts, tree barks, and other plant materials that containphenolic compounds. Most colored fruits, berries, and vegetables areknown to contain phenolic compounds. Examples of plants, fruits,berries, and vegetables that contain phenolic compounds include, but arenot limited to, blueberries, bilberries, elderberries, plums,blackberries, strawberries, red currants, black currants, cranberries,cherries, raspberries, grapes, currants, hibiscus flowers, bell peppers,beans, peas, red cabbage, purple corn, and violet sweet potatoes. Theraw plant material may be used either as is (wet) or may be dried priorto extraction. Optionally, the raw plant material may be presorted byseparating and removing the components low in anthocyanins andproanthocyanidins prior to extraction.

[0068] In one embodiment, the phenolic-enriched compositions of thepresent invention are obtained by extracting and purifying one or moreberries and/or fruits containing phenolic compounds including, but notlimited to, blueberries, bilberries, elderberries, plums, blackberries,strawberries, red currants, black currants, cranberries, cherries,raspberries, and grapes.

[0069] As used herein, the terms “phenols” and “phenolic compounds” areused interchangeably and include monomeric, oligomeric and polymericcompounds having one or more phenolic groups, and include, but are notlimited to, anthocyanins, proanthocyanidins, and flavonoids.

[0070] As used herein, the term “total phenol-enriched composition”refers to a composition enriched in one or more phenolic compounds andhaving substantially depleted levels of non-phenolic compounds presentin crude extracts of plants, fruits, berries, and vegetables. Examplesof such non-phenolic compounds include, but are not limited to, sugars,cellulose, pectin, amino acids, proteins, nucleic acids, plant sterols,fatty acids, and triglycerides.

[0071] The method of this invention is based on the discovery thatpurifying an extract containing phenols on a brominated polystyreneresin rather than on a conventional polystyrene resin or other resinsused in the art provides total phenol-enriched compositions havinghigher purities, as discussed below in detail.

[0072]FIG. 1 is a flowchart showing the steps of one embodiment of theprocess of this invention in which a composition enriched in totalphenols may be prepared. The process of this invention provides aneconomical and efficient method of obtaining compositions enriched intotal phenols by eliminating several process steps and by reducing theamount of reagents needed in the process, thereby reducing productioncosts and waste disposal issues.

[0073] In one embodiment of the process of this invention, asillustrated in steps 10-70 in FIG. 1, phenolic compounds (e.g.,proanthocyanidins and anthocyanins) and non-phenolic compounds areextracted from a fresh or dried plant material (step 10). Those skilledin the art will recognize that a variety of extraction methods areavailable in the literature, such as vat extraction, percolation,countercurrent extraction, etc. The particular method of extractionemployed is not essential to the process of the present invention. Thedegree of comminutation of the plant material prior to the extractionprocess should provide sufficient particulate surface area for theextraction solvent to contact.

[0074] In one embodiment of the process shown in FIG. 1, the extractionstep (step 10) is accomplished by placing fresh or dried plant materialin an appropriate amount of extraction solvent. In one embodiment, theextraction solvent comprises an acidified alcohol solution having about0-95% ethanol in water and a suitable acid in an amount of about 0-3%,more preferably about 0.006-0.012% by weight. In another embodiment, theextraction solvent comprises an acidified alcohol solution havingbetween about 0-100% methanol in water and between about 0-3% by weightof a suitable acid. Suitable acids that may be used in the extractionstep include, but are not limited to, sulfuric acid (H₂SO₄), acetic acid(HOAc) or hydrochloric acid (HCl). The addition of an acid to theextraction solvent prevents degradation of the proanthocyanidins andanthocyanins. Thus, in one embodiment the acidic conditions aremaintained throughout most of the steps of the process of this inventionas illustrated in FIG. 1. The plant material is contacted with theextraction solution for an appropriate amount of time at a temperaturebetween about room temperature and 75° C., preferably at 40° C., to formthe crude extract. The amount of plant material to extraction solventused in the extraction process varies between about 2:1 to about 1:20 ona gram to milliliter basis. In one embodiment, the ratio of plantmaterial to extraction solvent is between about 1:4 and 1:8.

[0075] The crude extract contains phenolic compounds such asproanthocyanidins, anthocyanins and other phenolics, as well asundesired non-phenolic materials such as sugars, pectin, plant sterols,fatty acids, triglycerides, and other compounds. Solid residue containedin the crude extract is separated from the liquid portion, and thesolids are either re-extracted as described above or discarded.

[0076] In one embodiment of step 10 (FIG. 1), pectinase is added eitherto the plant material or to the extraction solvent before or during theextraction process. Alternatively, the pectinase can be added to thecrude extract after the extraction process is complete. The pectinaseserves to prevent the extract from gelling at any point during or afterthe extraction process so that it will remain flowable during the columnpurification. The amount of pectinase added will depend, of course, onthe amount of plant material used to prepare the extract. Typically, thepectinase is added in an amount between about 0 and 0.12% by weight ofthe plant material.

[0077] With continued reference to FIG. 1, if either an ethanolic ormethanolic extraction solvent was used to prepare the crude extract instep 10, the crude extract is concentrated (step 20) until the crudeextract contains less than 6% ethanol or methanol, preferablymaintaining a temperature of 40° C. or less during concentration. Wateris added to dilute the concentrated crude extract, and the diluted crudeextract is either concentrated and diluted again with water prior tostep 30, or is carried on directly to step 30 without performing asecond dilution. Of course, if water was used as the extraction solutionin the preparation of the crude extract, step 20 is not necessary, andin this case the crude extract from step 10 is taken directly on to step30 as shown by the dashed arrow in FIG. 1.

[0078] Step 30 of the process shown in FIG. 1 comprises filtering thecrude extract from step 10 or 20 to remove solids that may haveprecipitated from the crude extract. The inventors discovered that byadjusting the extraction conditions as described for step 10, the amountof undesirable non-phenolic compounds that precipitate from the crudeextract by filtration in step 30 is increased. Various filtrationmethods may be employed in filtration step 30 of the process of thisinvention. One filtration method that may be employed in step 30comprises adding a measured amount of a filter aid such as diatomaceousearth or cellulose to the crude extract. The mixture of crude extractand filter aid is preferably shaken or stirred until homogeneous andfiltered through a bed of filter aid. The bed is washed with an aqueousacidic solution, preferably about 0.006% aqueous sulfuric acid.

[0079] Other filtration methods that may be used in step 30 of FIG. 1include filtering the crude extract through a bed of sand or a 30 micronpolypropylene filter that is preferably covered with glass wool. Yetanother filtration method comprises using a bag filter (a bag-shapedcloth filter composed of polyethylene or polypropylene), which mayadvantageously be placed in-line with the purification column of step 40described below. The filters described above are used to removeprecipitated solids and are not size exclusion filters.

[0080] To isolate the phenolic compounds according to the method shownin FIG. 1, the filtered extract isolated in step 30 is contacted with abrominated polystyrene adsorbent material capable of releasablyadsorbing the phenolic compounds such as proanthocyanidins andanthocyanins, but which retains less of the undesired non-phenolicmaterials that were present in the filtered extract. The presentinventors discovered that a high purity composition enriched in totalphenols could be obtained by purifying the filtered extract isolated instep 30 on a brominated polystyrene resin, such as SP-207 (Supelco;Bellafonte, Pa..), manufactured by Mitsubishi Chemical America. SP-207resin is a macroporous, brominated styrenic polymeric bead type resindesigned for reversed-phase chromatographic applications, and has aparticle size distribution between about 250-600 microns and a pore sizerange between about 100-300 Angstroms. The bromination of the aromaticrings provides increased hydrophobicity to the polystyrene resin, and isdesigned to provide a resin having increased selectivity for hydrophobicmolecules relative to conventional styrene-divinylbenzene polymericreversed-phase supports. Because of its tight binding properties,brominated polystyrene resin is not typically used in the purificationof natural products.

[0081] Thus, since it was known that conventional polystyrene resinstend to bind phenolic compounds such as proanthocyanidins andanthocyanins so tightly that it is very difficult to elute suchcompounds from the polystyrene resin, it was expected that thebrominated polystyrene resin would bind phenolic compounds even tighter.Therefore, it was not expected that a brominated polystyrene resin wouldbe suitable for the purification of phenolic compounds. However, theinventors surprisingly and unexpectedly discovered that the brominatedpolystyrene resin binds phenolic compounds such as proanthocyanidins andanthocyanins less tightly than non-brominated polystyrene resins, butstill allows for the separation of phenolic compounds from undesirednon-phenolic compounds.

[0082] In one embodiment of the method shown in FIG. 1, the filteredextract isolated in step 30 is loaded onto a column packed withbrominated polystyrene resin having a particle size distribution betweenabout 250-600 microns and a pore size range between about 100-300Angstroms (step 40). However, while step 40 is described herein in termsof contacting the extract with a resin packed into a column, such adescription is merely for ease of explanation. Thus, the resin need notbe packed into a column in order to perform the method of thisinvention. The amount of filtered extract that is loaded onto the columndepends on the plant material used to prepare the crude extract. Forexample, when the crude extract is prepared from bilberries, about 16-30grams of total phenols may be loaded per liter of resin. As anotherexample, when the crude extract is prepared from blueberries, about15-45 grams of total phenols may be loaded per liter of resin. When thecrude extract is prepared from elderberries, about 15-40 grams of totalphenols may be loaded per liter of resin. The filtered extract may bediluted with water prior to loading if the solids concentration in theconcentrated crude extract exceeds 200 grams per liter. The fractionseluting during column loading in step 40 (FIG. 1) are collected as“fraction 1.”

[0083] Subsequent to loading the filtered crude extract onto the resin,undesired non-phenolic materials (e.g., sugars, salts, organic acids,etc.) which have little or no affinity for the adsorbent are eluted fromthe resin with an aqueous wash solvent comprising at least 0.003% acidsuch as aqueous sulfuric acid, aqueous acetic acid or aqueoushydrochloric acid (FIG. 1, step 50). For example, about three columnvolumes of 0.006% aqueous sulfuric acid or 0.1% aqueous acetic acid canbe used to elute the extraneous materials. The eluent is collected as“fraction 2.”

[0084] With continued reference to FIG. 1, the column is next elutedwith a first eluent comprising a polar organic solvent such as about 50to 70% ethanol/water or about 50 to 90% methanol/water (step 60).Typically about 2 to 12 column volumes of eluting solvent are used inStep 60. In one embodiment, the first eluent contains about 0.003% of anacid such as sulfuric acid, hydrochloric acid or acetic acid. Thefraction(s) collected during elution step 60 are collected as “fraction3.” “Fraction 3” contains a portion of the phenolic compounds containedin the crude extract and is particularly enriched in anthocyanins andcontains proanthocyanidins.

[0085] After the majority of the anthocyanins have been eluted from thecolumn, as determined by UV-VIS spectroscopy, the column is eluted witha second eluent (step 70; FIG. 1) comprising a polar organic solventcomprising a greater percentage of ethanol or methanol than the solventused to elute the anthocyanins (step 60). For example, the second eluentmay comprise about 50 to 90% ethanol/water or about 75 to 100%methanol/water. The fraction(s) collected during elution step 70 arecollected as “fraction 4.” “Fraction 4” contains an additional portionof the phenolic compounds originally contained in the crude extract andis typically enriched in proanthocyanidins. “Fraction 4” may alsocontain anthocyanins not isolated during elution step 60.

[0086] Recovery of the phenolic compounds in “fraction 3” and “fraction4” can be accomplished in any convenient manner such as by evaporation,distillation, freeze-drying, and the like, to provide a totalphenol-enriched composition of this invention.

[0087] The above-described process is suitable for preparingcompositions sufficiently enriched in total phenols for use asnutraceuticals from a variety of plant materials that contain phenoliccompounds including, but not limited to, elderberries, plums,blueberries, bilberries, blackberries, strawberries, red currants, blackcurrants, cranberries, cherries, raspberries, grapes, hibiscus flowers,bell peppers, beans, peas, red cabbage, purple corn, and violet sweetpotatoes. In one embodiment, the enriched compositions of this inventioncontain at least 10-80% total phenols. In another embodiment, thecompositions contain at least 12% total phenols. In yet anotherembodiment, the compositions contain at least 25% total phenols.

[0088] It was discovered that the total phenol-enriched compositions,and in particular the compositions isolated from “fraction 3,” “fraction4,” or a combination thereof, prepared from fruits and berries inparticular produce similar HPLC chromatograms having the characteristicpeaks such as those shown in FIGS. 12 and 13 that are not contained inHPLC chromatograms of compositions prepared from plant material otherthan fruits and berries. For example, the HPLC chromatograms of alltotal phenol-enriched compositions prepared from fruits and berriesaccording to the method illustrated in FIG. 1 and isolated from“fraction 4” were found to contain characteristic peaks between 60 and75 minutes similar to peaks in the chromatogram shown in FIGS. 12 and 13for a “fraction 4” composition isolated from elderberries. The totalphenol-enriched compositions of this invention, isolated either from“fraction 3,” “fraction 4,” or a combination thereof, and preparedspecifically from fruits and berries have anti-infective (e.g.,antiviral) and anti-inflammatory activity, as described below in detail.

[0089] When the total phenol-enriched compositions of this invention areanalyzed by IR spectrometry, characteristic peaks from the phenoliccompounds are also observed. More specifically, the totalphenol-enriched compositions of this invention are characterized ashaving IR absorption peaks substantially as illustrated in FIGS. 33-40.

[0090] It was also discovered that the total phenol-enrichedcompositions (e.g., “fraction 3,” “fraction 4,” or a combinationthereof) could be further partitioned into a “polar”proanthocyanidin-enriched fraction and a “non-polar”proanthocyanidin-enriched fraction using low pressure Vacuum LiquidChromatography (VLC) on a reversed-phase lipophilic column, such as aC-18 column as described in detail in Example 11 and as shown in FIG.15. For example, a “fraction 3” composition isolated from an elderberryextract was dissolved in water and loaded onto a C-18 column. The columnwas washed with 100% water to collect materials that are not stronglyretained by the C-18 media. The flow through and Wash fractions Werecombined as “fraction 5” and contained the more polar proanthocyanidins.Thus, “fraction 5” is referred to herein as the “polar”proanthocyanidin-enriched fraction (FIG. 15). The polarproanthocyanidin-enriched “fraction 5” from elderberry typically hassome purple color, suggesting that the polymers in this fraction containat least one or more cationic anthocyanidin subunits within theoligomeric proanthocyanidin chains. The VLC column was then eluted with30 to 100% methanol to collect the proanthocyanidins that are morestrongly retained by the C-18 media used in the low-pressure column. Themethanol fractions were combined as “fraction 6” and containedproanthocyanidins that are less polar than those collected in “fraction5.” Thus, “fraction 6” is referred to herein as the “non-polar”proanthocyanidin-enriched fraction (FIG. 15). The non-polarproanthocyanidin-enriched “fraction 6” has little if any color,suggesting that the oligomeric proanthocyanidin chains in this fractiondo not contain cationic anthocyanidin subunits.

[0091] Thus, the present invention provides a method of convenientlyseparating the polar proanthocyanidins from the non-polarproanthocyanidins contained in either “fraction 3,” “fraction 4,” or acombination thereof. It was also found that a polarproanthocyanidin-enriched “fraction 5” and non-polarproanthocyanidin-enriched “fraction 6” could be isolated directly byloading a crude filtered aqueous extract (FIG. 1, step 30) onto a C-18VLC column. It is to be understood that the terms “polar” and“non-polar” when used to describe the isolated proanthocyanidin-enrichedfractions 5 and 6, respectively, refer to the polarity of theproanthocyanidins in fractions 5 and 6 relative to one another, that is,how the particular fractions behave on a C-18 VLC column. The polarproanthocyanidin-enriched compositions (fraction 5) and the non-polarproanthocyanidin-enriched compositions (“fraction 6”) of this inventionhave substantially reduced levels of anthocyanins, as discussed in theExamples.

[0092] The polar and non-polar proanthocyanidin-enriched fractions(“fraction 5” and “fraction 6,” respectively) were found to havedifferent biological activities, and the non-polar fraction was found tohave greater antiviral activity than the polar fraction in certainassays as described in Example 17.

[0093] Each of the polar and non-polar proanthocyanidin-enrichedfractions 5 and 6, respectively, can be purified further as shown inFIG. 15 and as described in Examples 12-14. For example, the polarproanthocyanidin-enriched “fraction 5” isolated during the VLCseparation can be loaded onto a semi-preparative C-18 HPLC column thatreleasably retains the polar proanthocyanidins. The column is thenwashed with a solvent gradient comprising increasing percentages ofacetonitrile, methanol or ethanol to elute most of the anthocyanins andother polar compounds, and then with at least 60% acetonitrile, methanolor ethanol to elute “fraction 7” containing the purified polarproanthocyanidins (FIG. 15). Additionally, the non-polarproanthocyanidin-enriched “fraction 6” isolated during the VLCseparation can be further purified by gel filtration or reversed-phasesemi-preparative HPLC. Gel filtration, also called size exclusion or gelpermeation chromatography, is a liquid chromatography technique thatseparates molecules according to their size. This type of media retainssmaller compounds, while the larger non-polar proanthocyanidin-enriched“fraction 8” (FIG. 15) elute with the flow-through eluent. The purifiedpolar and non-polar proanthocyanidin-enriched fractions 7 and 8,respectively, of this invention have substantially reduced levels ofanthocyanins and flavonoids, and also have substantially reduced levelsof non-phenolic compounds. It was further observed that the purifiedpolar and non-polar proanthocyanidin-enriched “fraction 7” and “fraction8”, respectively, have different biological activities.

[0094] The total phenol-enriched compositions (“fraction 3,” “fraction4,” or a combination thereof), polar proanthocyanidin-enrichedcompositions (fractions 5 and 7), and non-polarproanthocyanidin-enriched compositions (fractions 6 and 8) of thisinvention possess a range of biological activities. For example, thecompositions of this invention were found to have antiviral activities,as described in Examples 15 and 16. The compositions of this inventioncan be used either alone or in combination with other antiviral agentsto prevent and/or treat diseases induced by or complicated with viralinfections from viruses including, but not limited to, influenza A, B,and C, parainfluenza virus, adenovirus type 1, Punta Toro Virus A,Herpes simplex virus I and II, rhinovirus, West Nile virus,Varicella-zoster virus and measles virus. Accordingly, the totalphenol-enriched compositions, polar proanthocyanidin-enrichedcompositions, and non-polar proanthocyanidin-enriched compositions ofthis invention can be advantageously used in prophylactic andtherapeutic applications against diseases induced by such viruses byadministering a therapeutically effective amount of a composition ofthis invention.

[0095] Proanthocyanidins have also been investigated asanti-inflammatory substances due to their inhibition of cyclooxygenase(COX) activity. It has been shown that it is desirable foranti-inflammatory substances to be selective for COX-2 inhibition ratherthan COX-1 inhibition. Accordingly, another aspect of this inventioncomprises a method of treating inflammatory diseases in mammalscomprising administering a therapeutically effective amount of a totalphenol-enriched composition, polar proanthocyanidin-enrichedcomposition, or a non-polar proanthocyanidin-enriched composition ofthis invention. For example, total phenol-enriched compositions isolatedas fractions 3 and 4 during purification of a blueberry extract werefound to have high COX-2/COX-1 inhibition selectivity and an IC₅₀ of 108μg/mL (Example 17). The compositions of this invention can be usedeither alone or in combination with other anti-inflammatory agents toprevent or inhibit inflammatory responses. Such responses may be causedby conditions or diseases including, but not limited to, osteoarthritis,allergenic rhinitis, cardiovascular disease, upper respiratory diseases,wound infections, neuritis and hepatitis.

[0096] It is known that proanthocyanidins isolated from cranberries andblueberries inhibit bacteria from attaching to the bladder wall, therebyreducing the potential for maladies such as urinary tract infections(Howell, et al., New England J. Medicine, 339:1085-1086 (1998)). It hasbeen postulated that proanthocyanidins exert their effect by inhibitingthe adhesion of bacteria. Accordingly, another aspect of this inventioncomprises a method of preventing or treating urogenital infections in amammal comprising administering an effective amount of a totalphenol-enriched composition, polar proanthocyanidin-enrichedcomposition, or a non-polar proanthocyanidin-enriched composition ofthis invention in an amount sufficient to prevent, reduce, or eliminatethe symptoms associated with such infections. The compositions of thisinvention can be used either alone or in combination with otherantimicrobial agents.

[0097] It is further known that proanthocyanidins are potentantioxidants. For example, the antioxidant effects of proanthocyanidinsare presumed to account for many of their benefits on the cardiovascularand immune systems. Accordingly, the total phenol-enriched compositions,polar proanthocyanidin-enriched compositions, and non-polarproanthocyanidin-enriched compositions of this invention may be used asdietary supplements (e.g., dietary antioxidants) and for the treatmentof disorders in humans and mammals. For example, the compositions ofthis invention may be used for improving visual acuity and for treatingcirculatory disorders, diabetes, and ulcers.

[0098] The total phenol-enriched compositions, polarproanthocyanidin-enriched compositions, and non-polarproanthocyanidin-enriched compositions of this invention can also becombined with immunoactive agents, including but not limited to,arabinogalactan, species of Echinacea, vitamins, minerals,polysaccharides and astragalus.

[0099] The total phenol-enriched compositions, polarproanthocyanidin-enriched compositions, and non-polarproanthocyanidin-enriched compositions of this invention can also becombined with antimutagenic agents including, but not limited to, greentea extracts, catechins, epicatechins, epigallocatechins,gallocatechins, and flavonoids.

[0100] The total phenol-enriched compositions, polarproanthocyanidin-enriched compositions, and non-polarproanthocyanidin-enriched compositions of this invention may beformulated as pills, capsules, liquids, or tinctures. In formulatingcompositions according to this invention, a wide range of excipients maybe used, the nature of which will depend, of course, on the intendedmode of application of the composition. Examples of excipients includepreservatives, carriers, and buffering, thickening, suspending,stabilizing, wetting, emulsifying, coloring and flavoring agents, and inparticular carboxy vinyl polymers, propylene glycol, ethyl alcohol,water, cetyl alcohol, saturated vegetable triglycerides, fatty acidesters or propylene glycol, triethanolamine, glycerol, starch, sorbitol,carboxymethyl cellulose, lauryl sulphate, dicalcium phosphate, lecithin,etc.

[0101] The foregoing description is considered as illustrative only ofthe principles of the invention. Further, since numerous modificationsand changes will readily occur to those skilled in the art, it is notdesired to limit the invention to the exact construction and processshown as described above. Accordingly, all suitable modifications andequivalents may be resorted to falling within the scope of the inventionas defined by the claims that follow.

[0102] The words “comprise,” “comprising,” “include,” “including,” and“includes” when used in this specification and in the following claimsare intended to specify the presence of stated features, integers,components, or steps, but they do not preclude the presence or additionof one or more other features, integers, components, steps, or groupsthereof.

EXAMPLES Example 1 Purification of Bilberry Using a Water Extraction

[0103] Three extractions were performed on 1 kg of dried bilberry rawmaterial. The first extraction used 6 L of water and the other twoextractions used 4 L of water. All extractions were acidified withconcentrated sulfuric acid to an acid concentration of 5 g/L. There wasapproximately an 88% recovery of anthocyanins into the crude extract.Exactly 2.3 L of the crude extract were filtered through a 30 micronpolypropylene filter with a layer of glass wool over the filter. Theglass wool was changed once and the filter rinsed with deionized water.The final volume of the filtrate was 2.43 L with a 90.9% recovery ofanthocyanins in the filtrate.

[0104] A column was packed with brominated polystyrene resin SP-207(Supelco; Belefonte, Pa.) and equilibrated with 0.1% acetic acid. Thecolumn was loaded with 2.24 L of the filtrate at a solids concentrationof 29.8 g/L using a flow rate of 2.2 mL/min. The loading bleed was lessthan 0.9% of the loaded anthocyanins with an overall loss of 4.07% ofthe anthocyanins in the loading and first two column washes. There wasan 88.4% recovery of the anthocyanins in the elution step and ananthocyanins mass balance of 92.5%. A few hundred milliliters of elutionproduct were evaporated to dryness on a rotary evaporator and thenlyophilized. Final assay of the dried product was by standardspectrophotometric determination of absorbance at 535 nm against adelphinidin chloride standard (102 absorbance units/g/L at 1.0 cm). Theenriched composition contained 43% total anthocyanins by weight.

Example 2 Purification of Bilberry Using a 70% Ethanol Extraction

[0105] Dried bilberry raw material (667 g), assayed at 2.0%anthocyanins, was extracted by percolation using 70% ethanol/watercontaining 3% sulfuric acid by volume. The solids in the crude extractcontained 3.9% by weight total anthocyanins. One liter of the firstextraction volume was mixed with 100 mL deionized water and evaporatedin vacuo to about 460 mL to remove the alcohol. Deionized water (300 mL)was added to the mixture, and an additional 170 mL of liquid wereevaporated. Deionized water (210 mL) was added to make the final volume800 mL. To the aqueous mixture was added 150 g of Celite 512 (0.5 to 0.9g of Celite per gram of solids). The mixture was shaken untilhomogeneous. The Celite/extract mixture was poured over a 30 g bed ofdamp Celite 512 under vacuum. Upon completion of filtration, the bed waswashed with 1.20 L of 1% aqueous sulfuric acid in 200 mL increments. Thefiltrate volume was 1855 mL. To the filtrate was added 145 mL ofdeionized water to give a final volume of 2.0 L.

[0106] A portion of the filtrate (695 mL) was loaded at 2.2 mL/minute(1.3 mL/min/cm²) onto a column loaded with 170 mL brominated polystyreneresin (SP-207). This gave a load value of 17 g of anthocyanins per literof column media. The column was washed with one column volume of 0.1%aqueous acetic acid followed by 2.5 column volumes of 0.1% HOAc/10%ethanol/90% water. The column was then eluted with 10 column volumes of70% ethanol/water, and the 70% ethanol fractions were combined andconcentrated in vacuo at 60° C. and 50 mbar to provide a dark, dry,shiny amorphous solid (“fraction 3”). Final assay of the dried productwas by standard spectrophotometric determination of absorbance at 535 nmagainst a delphinidin chloride standard (102 absorbance units/g/L at 1.0cm). The enriched composition contained 32% total anthocyanins byweight.

[0107]FIGS. 2 and 3 are HPLC chromatograms at 510 nm and 280 nm,respectively, of a total phenol-enriched composition (“fraction 3”)prepared from bilberries according to the process of this invention.

[0108] Table 1 summarizes the percent of each anthocyanin in a typicalanthocyanin-enriched composition (“fraction 3”). TABLE 1 Identificationand content of each anthocyanin present in a bilberry “fraction 3”Elution Name order % composition Delphinidin-3-O-galactoside 1 3.3Delphinidin-3-O-glucoside 2 3.9 Cyanidin-3-O-galactoside 3 2.1Delphinidin-3-O-arabinoside 4 2.6 Cyanidin-3-O-glucoside 5 2.8Petunidin-3-O-galactoside 6 1.0 Petunidin-3-O-glucoside 7 2.5Cyanidin-3-O-arabinoside 8 1.7 Peonidin-3-O-galactoside 9 0.3Petunidin-3-O-arabinoside 10 0.8 Malvidin-3-O-galactoside 11 2.1Peonidin-3-O-glucoside (co-elute) Malvidin-3-O-glucoside 12 2.5Peonidin-3-O-arabinose 13 0.1 Malvidin-3-O-arabinose 14 0.6 Total 26.3

Example 3 Total Phenol-Enriched Compositions from Blueberries

[0109] To 940 g of dried and ground blueberry (Van DrunenFutureCeuticals; Momence, Ill.) were added 4.0 liters of extractionsolvent (1.0% w/v sulfuric acid in 70% ethanol) in a 10 L round bottomflask. The flask was rotated in a constant temperature water bath heldat 40° C. for two hours. The mixture was swirled and filtered through a150 g bed of Celite 512 under vacuum. The blueberry biomass cake waswashed with 500 mL of extraction solvent. The cake was carefully scrapedaway from the Celite bed, poured into a round bottom flask, andre-extracted following the above-described procedure. A third extractionwas then performed. The three crude extracts were combined.

[0110] A portion of the combined extracts (2.00 L) was concentrated invacuo to 175 mL at a water bath temperature of 40° C. The evaporatedextract was diluted with deionized water to give 675 mL of crudeblueberry extract. The crude extract was loaded without filtration ontoa previously conditioned (i.e., washed with acetone) and equilibratedcolumn loaded with 170 mL of brominated polystyrene resin (SP-207). Thecolumn was washed with 0.1% acetic acid and with 0.1% HOAc/10% ethanol.The anthocyanins were eluted with 70% ethanol. The product pool wasevaporated in vacuo at 60° C. and 50 mbar. Final product assay was bystandard spectrophotometric determination of absorbance at 535 nmagainst a delphinidin chloride standard (102 absorbance units/g/L at 1.0cm). The purified blueberry composition (“fraction 3”) contained 18%total anthocyanins by weight, with an overall recovery of anthocyaninsof 95%.

[0111]FIGS. 4 and 5 are HPLC chromatograms at 510 nm and 280 nm,respectively, of a total phenol-enriched composition (“fraction 3”)prepared from blueberries according to the method of this invention.

Example 4 Higher Purity Total Phenol-Enriched Composition fromBlueberries

[0112] In this example, a portion of a total phenol-enriched compositionprepared from blueberries and having 18% total anthocyanins by weight,prepared as described in Example 3, was passed through either a strongor a weak anion exchange resin to remove residual acids in order toincrease the purity of the enriched composition.

[0113] Approximately 1.0 g of the total phenol-enriched blueberrycomposition was dissolved in 50 mL of water and passed through a 9 mLcolumn containing either a strong anion exchange resin (Super Q-650 M;TosoHaas; Montgomery, Pa.) or a weak anion exchange resin (DEM-63;Whatman). The column was washed with 30-35 mL of water. In the case ofthe strong anion exchange resin column, the resin was further washedwith 25 mL of 20% ethanol, followed by 40% ethanol. The compositionisolated from the strong anion exchange column contained 28.3% totalanthocyanins by weight, and the recovery was 88%. The compositionisolated from the weak anion exchange column contained 30.6% totalanthocyanins by weight, and the recovery was 88%.

Example 5 Total Phenol-Enriched Compositions from Bilberry UsingPectinase Treatment

[0114] Warm water (548 g) was added to 1024 g of frozen bilberries. Themixture was pureed in a blender and then heated to 40° C. Next, 150 μLof pectinase (Quest Super 7x; Quest International, Norwich, N.Y.) wereadded for a 30 minute treatment at 40° C. with stirring. Approximately 4mL of concentrated sulfuric acid were added to the slurry to achieve anacid concentration of 0.5% (w/w). The mixture was then heated to 45° C.and extracted for 15 minutes with very slow stirring. Dicalite (164 g)was added to the extracted mixture, which was then filtered over a 26 gDicalite bed. The resulting cake was washed three times with 400 mL ofwarm 0.1 % aqueous sulfuric acid. This extract was filtered through a 25μm pressure filter. All of the filtered extract (2.4 L) was loaded ontoa 170 mL SP-207 column. After loading, the column was washed with 0.1%aqueous acetic acid and eluted with 70% aqueous ethanol to provide“fraction 3”. “Fraction 3” was evaporated to dryness and then placed ona lyophilizer for 48 hours. The final product was assayed for totalanthocyanins by standard spectrophotometric determination of absorbanceat 535 nm. The total phenol-enriched composition contained 40% totalanthocyanins by weight. The overall recovery of anthocyanins wasapproximately 79%.

Example 6 Enriched Compositions from Elderberry Biomass Powder

[0115] Approximately 190 g of dried elderberry biomass powder (BINutraceuticals, Long Beach, Calif.) assayed at 1.88% anthocyanins and5.31 % total phenols were added to 1000 g of warm water. The solutionwas mixed thoroughly and transferred to a hot water bath at 45° C. Tothe solution was added 190 μL of pectinase (Super 7X, Quest), and thenthe mixture was allowed to sit for 30 minutes. The mixture was acidifiedto a pH of 2.5 using 2.5 mL of concentrated H₂SO₄ and gently mixed forten minutes. To this acidified mixture was added 164 g of Celite, andthen the acidified mixture was filtered over a 26 g Celite bed. Thefilter cake was washed three times with 400 mL of acidified warm water,for a total of 1200 mL. The filtrate was then filtered through a 25 μmpressure filter to provide an elderberry extract.

[0116] The elderberry extract was loaded onto 170 mL of SP-207(Mitsubishi Chemical) brominated polystyrene column at a rate of 2.3mL/min (1.3 mL/min/cm²). The eluent collected off the column duringloading was collected as “fraction 1.” After loading, the column waswashed with 3 column volumes (3×170 mL) of 0.006% aqueous sulfuric acid.The eluent from this wash was collected as “fraction 2.” The column wasthen eluted with 8-10 column volumes of 70% aqueous ethanol, which werecollected as “fraction 3.” The column was then washed with 3 columnvolumes of 90% aqueous ethanol, which were collected as “fraction 4.”The column was re-equilibrated with 8 column volumes of 0.006% aqueoussulfuric acid. Fractions 3 and 4 were evaporated to dryness and thenlyophilized until dry. Several of the fractions isolated during elutionfrom the brominated polystyrene resin were analyzed for anthocyanins andtotal phenols as described in Examples 7 and 8. Table 2 summarizes thecolumn data. TABLE 2 Analysis and recovery of anthocyanins andpolyphenols in elderberry fractions Anthocyanins Polyphenols % Purity %Recovery % Purity % Recovery “fraction 1” 0.05 2.79 1.37 24.4 “fraction2” 1.68 7.79 5.57 8.5 “fraction 3” 18.7 99.4 42.8 74.7 “fraction 4” 0.670.49 2.61 0.6

[0117] FIGS. 6-13 show the HPLC chromatograms of the filtered elderberryextract and of certain fractions isolated during column purification.The HPLC conditions used are those described in Example 9.

[0118]FIGS. 6 and 7 show the HPLC chromatograms at 280 nm and 510 nm,respectively, of the filtered elderberry extract.

[0119]FIGS. 8 and 9 show the HPLC chromatograms at 280 nm and 510 nm,respectively, of “fraction 1” collected during column loading of thefiltered elderberry extract onto the brominated polystyrene resin.

[0120]FIGS. 10 and 11 show the HPLC chromatograms at 280 nm and 510 nm,respectively, of “fraction 3” collected during column elution of thefiltered elderberry extract using 70% ethanol from the brominatedpolystyrene resin.

[0121]FIGS. 12 and 13 show the HPLC chromatograms at 280 nm and 510 nm,respectively, of “fraction 4” collected during column elution of thefiltered elderberry extract using 90% ethanol from the brominatedpolystyrene resin.

[0122] The total phenol-enriched compositions of this invention comprisethe compounds showing peaks in the region between 60 and 75 minutes inthe standard HPLC chromatograms substantially as shown in FIGS. 10-13.

Example 7 Quantitative Determination of Anthocyanins

[0123] This method is used to determine the total anthocyanins invarious biomass samples and dried purified total phenol-enrichedcompositions by UV-VIS spectrophotometry, using an external standard.Each sample tested (e.g., a concentrated total phenol-enrichedcomposition, dried biomass, or fresh/frozen biomass) requires adifferent preparation procedure as described below.

[0124] Total phenol-enriched compositions—Accurately weigh 75-100 mg ofthe purified total phenol-enriched composition into a 100 mL volumetricflask and dilute to volume with 2% HCl/MeOH. Mix well and dilute0.40-1.6 mL of this sample to 10.0 mL with 2% HCl/MeOH.

[0125] Dry Biomass—Into a coffee grinder place an amount of dry biomasssufficient to cover the blades of the grinder. Grind for about 1 minuteor until finely ground. Alternatively use a mortar and pestle to finelygrind the raw material. Accurately weigh about 50-100 mg of finelyground biomass into a 100 mL volumetric flask and then add about 80 mLof 2% HCl/MeOH and cap. Place the flask into a 50° C. oil bath or forcedair oven for 30-60 minutes, shake gently for 30 seconds, and sonicatefor 5 minutes. Allow the solution to cool to room temperature. Add 2%HCl/MeOH to the mark and mix. Filter a portion of the sample through a0.45 μm PTFE syringe filter into a vial. Dilute 1.0 mL of the filtrateto 10.0 mL with 2% HCl/MeOH. The dilution factor would be 10 mL/1 mL or10.

[0126] Frozen/Fresh Biomass—Weigh 400.0 g frozen/fresh biomass into a1000 mL polypropylene beaker. Add 400 g of near boiling water into thebeaker. Puree using a mechanical blender (Waring or other). Using awide-bore polyethylene dropper, remove a representative 0.5-1.5 g sampleand transfer into a tared 100 mL volumetric flask. Add 80 mL of 2%HCl/MeOH and cap. Place the flask into a 50° C. oil bath or forced airoven for 60-120 minutes, shake gently for 30 seconds and then sonicatefor 5 minutes. Allow the solution to cool to room temperature. Add 2%HCl/MeOH to the mark and mix. Filter a portion through a 0.45 μm PTFEsyringe filter into a vial. The dilution factor would be the totalweight of the biomass and water divided by the weight of the biomass[e.g., (400 g+400 g)/400 g=2].

[0127] Loss on Drying—The calculation to obtain the total anthocyaninscontent in the above samples requires the determination of the moisturecontent, or % LOD (loss on drying), of the material. To determine the %LOD, transfer and distribute evenly 0.5-3.0 g of sample into anaccurately weighed aluminum weigh pan, and record the weight to thenearest 0.1 mg. Place the sample in an oven at 105° C.±3° C. for 2 hours(do not exceed 2 hrs 15 min). After the sample has cooled to roomtemperature (a dessicator may be used), weigh the sample and record theweight to the nearest 0.1 mg. The % LOD is determined to the nearest 0.1% using Equation 1: $\begin{matrix}{{\% \quad {LOD}} = {1 - {\frac{W_{D} - W_{P}}{W_{SP} - W_{P}} \times 100}}} & {{Eq}.\quad 1}\end{matrix}$

[0128] where % LOD=percentage loss on drying; W_(D)=dry weight of thepan and sample (g); W_(P)=weight of the pan (g); and W_(SP)=initialweight of the pan and sample (g).

[0129] Assay Procedures—The UV/VIS spectrophotometer is set to read inphotometry mode with the visible lamp on. The instrument is zeroed at535 nm using 2% HCl/MeOH in a 1 cm pathlength glass, quartz, ordisposable polystyrene cuvette. The absorbance of the prepared sample ismeasured at 535 nm in the same or matched 1 cm cuvettes.

[0130] Calculations—The concentration of total anthocyanins iscalculated as shown in Equation 2: $\begin{matrix}{C_{ANTHOS} = \frac{{ABS}_{SAMP} \times {DF}}{E_{s}}} & {{Eq}.\quad 2}\end{matrix}$

[0131] where C_(ANTHOS)=concentration of the total anthocyanins in thesample (mg/mL); ABS_(SAMP)=absorbance of the sample at 535 nm;DF=dilution factor, as described below; and Es=absorptivity (absorbanceof a 1 mg/mL solution at 535 nm in 2% HCl/MeOH using a 1 cm cuvette) ofthe appropriate external standard, either cyanidin chloride (101.1; forcherry, cranberry, elderberry, and plum) or delphinidin chloride (102.0;for bilberry and blueberry). The dilution factor (DF) for a dry biomassis 1, and the dilution factor for fresh/frozen biomass is the totalweight of the biomass and water divided by the weight of the biomass(e.g., (400 g+400 g)/400 g). The dilution factor for a purified extractis the final dilution volume divided by the volume of the extractsolution (e.g., 10 mL/0.40 mL).

[0132] The percent total anthocyanins is calculated as shown in Equation3: $\begin{matrix}{{\% \quad {Anthos}} = \frac{C_{ANTHOS} \times {Volume} \times 100}{{Ws} \times S_{LOD}}} & {{Eq}.\quad 3}\end{matrix}$

[0133] where % Anthos=percentage of total anthocyanins in the sample;C_(ANTHOS)=concentration of total anthocyanins (mg/mL); Volume=initialvolume of the sample preparation (usually 100 mL); Ws=weight of thebiomass or total phenol-enriched compositions used in the preparation(usually 50-100 mg for dry biomass, 500-1500 mg for fresh/frozenbiomass, or 75-100 mg for purified extracts); and S_(LOD)=[(100- %LOD)/100] for dry or fresh biomass or purified extract (for fresh orfrozen biomass this factor does not apply).

Example 8 Quantitative Determination of Total Polyphenols

[0134] This method is used to quantitatively determine the totalpolyphenols in various biomass samples and dried purified enrichedcompositions by UV-VIS spectrophotometry, using gallic acid as theexternal standard.

[0135] The procedure requires a 20% Na₂CO₃ solution and 2% HCl/MeOH. Toprepare the Na₂CO₃ solution, weigh approximately 100 g of Na₂CO₃ into a500 mL volumetric flask containing about 350 mL deionized water.Sonicate for 10 minutes; shake to mix. Dilute to volume using deionizedwater and agitate until homogeneous. To prepare the 2% HCl/MeOH,transfer about 350 mL of methanol into a 500 mL volumetric flask. Pipetinto the flask 10.0 mL of HCl. Dilute to volume using methanol and mixuntil homogeneous.

[0136] To prepare the gallic acid stock standard, accurately weigh 100mg of gallic acid (Sigma; St. Louis, Mo.) into a 100 mL volumetricflask. Add 70 mL of deionized water and sonicate for 5 minutes untildissolved. Dilute to volume using deionized water, cap, and mix untilhomogeneous.

[0137] Each sample tested (e.g., total phenol-enriched composition, drybiomass, or fresh/frozen biomass) requires a different preparationprocedure and was prepared as described in Example 7.

[0138] Loss on Drying—The calculation to obtain the total polyphenolscontent in the above samples requires the determination of the moisturecontent, or % LOD, of the material. To determine the % LOD, transfer anddistribute evenly 0.5-3.0 g of sample into an accurately weighedaluminum weigh pan, and record the weight to the nearest 0.1 mg. Placethe sample in an oven at 105° C.±3° C. for 2 hours (do not exceed 2 hrs15 min). After the sample has cooled to room temperature (a dessicatormay be used), weigh the sample and record the weight to the nearest 0.1mg. The % LOD is determined to the nearest 0.1 % using Equation 1 above.

[0139] Colorimetric Development Procedures—A clean 100 mL volumetricflask is set aside to serve as the reagent blank. Two 100 mL volumetricflasks are labeled “high” standard and “low” standard. Using the gallicacid stock solution, pipet 800 μL into the “high” standard flask and 200μL into the “low” standard flask. For dry biomass samples, pipet 20 mLof the filtered solution into a 100 mL volumetric flask. Forfresh/frozen biomass samples, pipet 10 mL of the filtered solution intoa 100 mL volumetric flask. For purified samples, pipet 0.80-2.0 mL intoa 100 mL volumetric flask. The following are added to each of thevolumetric flasks (including the reagent blank) prepared above:

[0140] 1. Add sufficient deionized water to each flask to bring thetotal volume to approximately 65 mL.

[0141] 2. Pipet 5.0 mL of the FC Phenol Reagent (Sigma) into each flask,agitate gently.

[0142] 3. Pipet 15±2 mL of the 20% Na₂CO₃ solution into each flask.

[0143] 4. Mix the solutions in each flask with gentle swirling, diluteto volume with deionized water, cap, and invert.

[0144] 5. Allow the solutions to develop for at least three but not morethan four hours.

[0145] 6. Filter 10 mL aliquots of samples requiring filtration through0.45 μm PVDF syringe filters into suitable containers.

[0146] Assay Procedure—The UV-VIS spectrophotometer is set to read inphotometry mode with the visible lamp on. The analysis is carried out in1 cm pathlength glass, quartz, or disposable polystyrene cuvettes. Theinstrument is zeroed at 760 nm using the reagent blank. The absorbanceof each solution is measured at 760 nm in the same or matched 1 cmcuvettes.

[0147] Calculations—To calculate the concentration of total polyphenolsthe absorptivity of gallic acid must first be determined. This value isobtained as described in Equation 4: $\begin{matrix}{E_{R} = \frac{A_{R} \times D_{R}}{C_{R} \times \left( {1 - E_{LOD}} \right)}} & {{Eq}.\quad 4}\end{matrix}$

[0148] where E_(R)=absorptivity of the reference standard (gallic acid)at 760 nm in absorbance units/g/L; A_(R)=absorbance of the referencestandard solution; C_(R)=concentration of gallic acid in the stockstandard solution, D_(R)=dilution factor for the gallic acid standard(125 for “high” standard or 500 for “low” standard); and E_(LOD)=loss ondrying of the gallic acid solids as a percent.

[0149] The absorptivities for the “high” and “low” standards areaveraged for use in Equation 5 below. The concentration of totalpolyphenols in the color development sample preparations is calculatedas shown in Equation 5: $\begin{matrix}{C_{p} = \frac{A_{S} \times D_{FC}}{E_{R}}} & {{Eq}.\quad 5}\end{matrix}$

[0150] where C_(P)=concentration of total polyphenols in the FC samplepreparation (mg/mL); A_(S)=absorbance of the FC sample preparation;D_(FC)=sample dilution factor, where DF is typically 5 for dry biomass,10 for fresh/frozen biomass, and 50-125 for purified enrichedcomposition; and E_(R)=average absorptivity of the gallic acidstandards.

[0151] The percent total polyphenols is calculated as shown in Equation6: $\begin{matrix}{{\% \quad P} = \frac{C_{P} \times V_{S} \times D_{S} \times 100}{W_{S} \times S_{LOD}}} & {{Eq}.\quad 6}\end{matrix}$

[0152] where % P=percentage of total polyphenols in the sample;C_(P)=the concentration of total polyphenols (mg/mL); V_(S)=volume oforiginal sample preparation (usually 100 mL); W_(S)=weight of thebiomass or purified composition used in the original sample preparation(usually 50-100 mg for dry biomass, 500-1500 mg for fresh/frozenbiomass, and 75-100 mg for purified extracts); D_(S)=original sampledilution factor, where D_(S) is 1 for dry biomass, 2 for fresh/frozenbiomass, or 1 for purified extract; and S_(LOD)=[(100-% LOD)/100] forbiomass or purified extracts. For fresh or frozen biomass this factordoes not apply.

Example 9 HPLC Qualitative Assay

[0153] This method is used to qualify compounds in various biomasses andpurified enriched compositions by high performance liquid chromatography(HPLC). Each type of sample requires a different preparation procedureas described below.

[0154] Dry Biomass: The dry biomass, if not already powdered, is groundthrough a 1 mm screen using the Wiley mill. Using an appropriately sizedextraction thimble and a soxhlet extraction apparatus, weigh outapproximately 12 g of powdered biomass into the thimble and extractusing 200 mL of methanol. Extract through at least 20 cycles or untilthe extraction solvent is clear. Transfer the extract quantitatively toa 250 mL volumetric flask using methanol, dilute to volume and mix.Filter the extract through a 0.45 μm PTFE syringe filter into an HPLCvial.

[0155] Frozen/Fresh Biomass: Weigh 400 g frozen/fresh biomass into a1000 mL polypropylene beaker. Add 400 g of near boiling water into thebeaker. Puree using a mechanical blender (Waring or other). Using awide-bore polyethylene dropper, remove a representative 0.5-1.5 g sampleand transfer into a tared 100 mL volumetric flask. Add 80 mL MeOH, cap,and heat at 50° C. for 30 minutes. Allow the solution to cool to roomtemperature, adjust to volume with methanol, and then sonicate untilhomogeneous. Filter a portion through a 0.45 μm PTFE syringe filter intoan HPLC vial.

[0156] Purified Enriched Composition: Accurately weigh 50-100 mg of theenriched composition into a glass scintillation vial and add 10.0 mL of50% MeOH/H₂O. Sonicate for 5 minutes. Filter through a 0.45 μm PTFEsyringe filter into an HPLC vial.

[0157] The HPLC is set up as required. In one embodiment of thisinvention, the aqueous mobile phase was prepared by mixing 5 mL oftrifluoroacetic acid (TFA) into 1000 mL of high purity, Type 1 water. A20 μL sample was injected at ambient temperature. A 280 nm wavelengthwas used for detection, the flow rate was 1.0 mL/min, and the run timewas 105 minutes. A Zorbax column was packed with 5 μm SBC-18 in a150×4.6 mm ID column. In this embodiment, the mobile phase was set up asfollows: channel A: 100% acetonitrile; channel B: 0.5% TFA in H₂O; andchannel C: 100% methanol. Table 3 summarizes the HPLC gradient for thisembodiment of the invention.

[0158] If available, standard preparations of compounds known to existin the sample may be prepared at concentrations of approximately 1mg/mL. These standard preparations can be used to determine theapproximate retention times and thus identify those compounds in thesample chromatograms. As this method is used for qualification purposesonly, no calculations are required. TABLE 3 HPLC gradient forqualitative analysis Time (min) % A % B % C 0.0 0 95 5 7.0 5 90 5 32.1 884 8 33.0 9 83 8 63.0 14 78 8 91.5 27 65 8 99.0 72 20 8 104.0 72 20 8104.1 0 95 5 112.0 0 95 5

Example 10 Quantitative HPLC Method for Determination of PercentProanthocyanidins

[0159] This HPLC method is used to determine the amount ofproanthocyanidins in various fractions and enriched compositions. Eachtype of sample requires a different preparation and is prepared asdescribed in Example 9. The method uses a 5 μm Zorbax column packed withStablebond SBC-18 in a 150×4.6 mm column. The flow rate was 1.5 mL/min,the detector was set at 280 nm, the injection volume was 10 μL, and therun time was 24 min. The mobile phase was: channel A=100% acetonitrile;channel B=0.1% trifluoroacetic acid in water; channel C=100% methanol.The gradient employed is provided in Table 4. The proanthocyanidinstypically eluted as a group of broad peaks in the HPLC chromatogram atelution times between 11-22 minutes. TABLE 4 HPLC gradient for %analysis for proanthocyanidins Time (min.) % A % B % C 0 14 78 8 9 14 788 17 34 58 8 22 34 58 8 22.1 14 78 8 26 14 78 8

[0160] To quantitate the proanthocyanidins, a previously preparedin-house proanthocyanidin standard is utilized with a purity greaterthan 90%. A sample of this is prepared at 5.5mg/mL in 70% ethanol andanalyzed using the HPLC method described in this Example. Thechromatogram for this standard includes a large, broad peak in the 11-22minute retention time range (as seen in FIG. 14) which is due to theproanthocyanidins. Manually integrate the entire 11-22 minute peak. Thepeak area response factor for the standard is then determined bydividing the entire 11-22 minute peak area by the product of thestandard's concentration and its purity as shown in Equation 7:$\begin{matrix}{{RF} = \frac{PA}{C_{std} \times P_{std}}} & {{Eq}.\quad 7}\end{matrix}$

[0161] where RF=peak area response factor for the standard (areaunits/mg/mL); PA=peak area of the proanthocyanidins in the standard;C_(std)=concentration of the standard solution in mg/mL; andP_(std)=standard purity as a percent (usually 0.90).

[0162] The percent proanthocyanidins in a sample can be determined usingthe sample preparation and HPLC analysis method described above. Thetotal peak area in the 11-22 minute retention time range is determinedfor the sample in question. Before any calculation can be made, however,the peak areas of non-proanthocyanidin compounds in the proanthocyanidinretention time range must be subtracted from the overall total peakarea. Non-proanthocyanidin compounds often appear as sharp peaksco-eluting with or on top of the broad proanthocyanidins' peak, andtheir UV spectrum by diode array is often different from the bulk of theproanthocyanidin peak. To determine the peak area ofnon-proanthocyanidin peaks, manually integrate these peaks, total theirpeak area and subtract this area from the total 11-22 minute peak area.Once the net area of the proanthocyanidins' peak in the sample has beendetermined, divide this value by the peak area response factor for thein-house standard to obtain the concentration of proanthocyanidins inthe sample as shown in Equation 8: $\begin{matrix}{C_{proanthos} = \frac{{PA}_{samp} \times {DF}}{RF}} & {{Eq}.\quad 8}\end{matrix}$

[0163] where C_(proanthos)=concentration of total proanthocyanidins inthe sample (mg/mL); PA_(samp)=corrected total peak area for the sample;DF=dilution factor (1 for dry biomass, 2 for fresh/frozen biomass, and 1for an enriched composition); and RF=peak area response factorcalculated using Equation 7.

[0164] The percent total proanthocyanidins is calculated as shown inEquation 9: $\begin{matrix}{{\% \quad {Proanthocyanidins}} = \frac{C_{proanthos} \times V \times 100}{W_{s}}} & {{Eq}.\quad 9}\end{matrix}$

[0165] where % Proanthocyanidins=percent of total proanthocyanidins inthe sample; C_(proanthos)=concentration of total proanthocyanidins(mg/mL); V=volume of the sample preparation (usually 250 mL for drybiomass, 100 mL for fresh/frozen biomass, or 10 mL for enrichedcompositions); and W_(s)=weight of the biomass or enriched compositionused in the sample preparation (usually 12,000 mg for dry biomass,500-1500 mg for fresh/frozen biomass, or 50-100 mg for enrichedcompositions).

Example 11 Partitioning Polar and Non-Polar Proanthocyanidins Directlyfrom a Filtered Elderberry Extract

[0166] In this example, a filtered elderberry extract was prepared and,rather than being purified on a brominated polystyrene resin, wasinstead loaded directly onto a vacuum liquid chromatography (VLC) columnto partition polar proanthocyanidins and non-polar proanthocyanidinsdirectly from a filtered extract according to the method illustrated inFIG. 15.

[0167] A 50 mL C-18 VLC column was prepared by filtering a 50 mL slurryof Bakerbond 40 μm flash chromatography C-18 media in methanol through a60 mL fritted glass filter. The column was conditioned by washing withmethanol and then with water. A 300 mL portion of the filteredelderberry extract, containing 12.0 g of solids, 74 mg of anthocyaninsand about 780 mg of proanthocyanidins, was loaded onto the column. AnHPLC chromatogram of the filtered extract using the HPLC methoddescribed in Example 10 is shown in FIG. 16. The flow-through eluent(about 300 mL) and a 100 mL wash (0.1% trifluoroacetic acid (TFA)) werecombined to provide the polar proanthocyanidin “fraction 5.” An HPLCchromatogram at 280 nm of “fraction 5” is shown in FIG. 17. The columnwas then eluted with 100 mL each of 30, 40, 50, 60, 70, and 100%methanol containing 0.1% TFA. An HPLC chromatogram at 280 nm of thenon-polar proanthocyanidin “fraction 6” isolated in the 60% methanoleluent is shown in FIG. 18. The fractions were assayed for anthocyaninsand proanthocyanidins by the methods described in Examples 7 and 10.Table 5 summarizes the results for this experiment. TABLE 5 Partitioningof Elderberry % Proantho- Anthocyanins Proanthocyanidins cyanidinElution Fraction (mg) (mg) purity Flow-Through + 54 554 4.7 Wash  30%MeOH 11 10 1.1  40% MeOH 3 20 12  50% MeOH 1 92 92  60% MeOH 0.2 46 92 70% MeOH 0.1 41 100 100% MeOH N/A 21

[0168] The results indicate that 71% (558 mg) of the proanthocyanidinsin the filtered extract were collected during the loading and wash.These proanthocyanidins were the more polar proanthocyanidins. Thenon-polar proanthocyanidins eluted when the methanol concentration wasincreased to at least 40%. The purity of the proanthocyanidins elutingin the 50-70% methanol fractions was high due to the fact that themajority of the solids contained in the filtered elderberry extracteluted in the loading eluent, water wash, and 30% methanol wash.

Example 12 Partitioning Elderberry Proanthocyanidins by VLC Followed byPurification by Gel Permeation Chromatography or Semi-Preparative HPLC

[0169] A total phenol-enriched composition was prepared from elderberrydried biomass (Martin Bauer; Germany) by collecting the 70% ethanolfraction (“fraction 3”) during elution from a brominated polystyreneresin using the procedure as described in Example 6. A portion (2.00 g)of this total phenol-enriched composition was dissolved in 50 mL ofwater and loaded onto a 15 mL C-18 VLC column prepared with Bakerbond 40μm C-18 media. The flow-through eluent and the 25 mL water wash werecombined and freeze-dried, yielding 733 mg of the polarproanthocyanidins fraction (“fraction 5”). The column was then washedwith 25 mL of 50% methanol. The non-polar proanthocyanidins (“fraction6”) were eluted with 25 mL of 70% methanol. The methanol in thisfraction was removed and the resulting water suspension wasfreeze-dried, yielding 192 mg of the non-polar proanthocyanidin fraction(“fraction 6”), which by HPLC assay was 100% proanthocyanidins. Thisfraction had little if any color, suggesting that the oligomericproanthocyanidins chains in this fraction do not contain cationicanthocyanin units.

[0170] The polar proanthocyanidins fraction (“fraction 5”) was furtherpurified by semi-preparative HPLC to remove residual anthocyanins andother more polar impurities. The conditions for the semi-preparativeHPLC purification of these solids are described below.

[0171] The semi-preparative HPLC method used a 2.5×10 cm Waters PrepPakcartridge filled with 6 μm, 60 Angstrom, Nova-Pak HR C-18 media (Waters;Milford, Mass.). The mobile phase was: channel A=100% acetonitrile;channel B=0.1% trifluoroacetic acid; channel C=100% methanol. Thegradient employed in this embodiment was as provided in Table 6. Theflow rate was 30 mL/min, the detector was set at 280 nm, and theinjection volume was typically 3-5 mL of a solution containing 50-125 mgof solids. The run time was 30 minutes. The proanthocyanidins werecollected in a broad peak that eluted between 13-20 minutes. TABLE 6HPLC gradient for Elderberry proanthocyanidin purification Time (min.) %A % B % C 0.0 11 81 8 11.0 11 81 8 19.0 34 58 8 24.0 34 58 8 25.0 82 108 30.0 82 10 8 30.1 11 81 8

[0172] About 600 mg of the polar proanthocyanidins fraction (“fraction5”) were dissolved in 25 mL of water. Approximately 3 mL (75 mg) wereinjected in each of eight runs. The proanthocyanidin peaks elutingbetween about 12-18 minutes in each run were collected, pooled, andevaporated on a rotary evaporator, and the residual aqueous solutionfreeze-dried. Approximately 100 mg of purified polar elderberryproanthocyanidins (“fraction 7”) were obtained from 600 mg of the polarproanthocyanidin solids (“fraction 5”) after VLC separation. An HPLCchromatogram at 280 nm for the VLC-isolated polar proanthocyanidinsafter semi-preparative HPLC purification is shown in FIG. 19. The polarfront, comprising sugars, amino acids, anthocyanins, organic acids, andsmall flavonoid compounds, was removed by the semi-preparative HPLCpurification, as evidenced by the absence of these peaks in FIG. 19. A¹³C NMR spectrum of the purified polar proanthocyanidins (“fraction 7”)is shown in FIG. 20.

[0173] The non-polar proanthocyanidins fraction (“fraction 6”) wasfurther purified by gel filtration chromatography. A portion (48 mg) ofthe non-polar proanthocyanidin fraction (“fraction 6”) isolated duringthe VLC separation was dissolved in 20 mL of warm water and loaded ontoa 14 mL Sephadex LH-20 column that had previously been equilibrated withwater. The loading eluent was collected and combined with a 40 mL columnwater wash. Most of the non-polar proanthocyanidins eluted from thecolumn at this point while most of the smaller flavonoid impurities wereretained. The combined loading and wash eluents were freeze-dried toprovide 32 mg of the purified non-polar proanthocyanidins “fraction 8.”These solids possessed strong antiviral activity. FIGS. 21 and 23 showthe HPLC chromatograms at 280 nm and 368 nm, respectively, of thenon-polar proanthocyanidins (“fraction 6”) before the Sephadex LH-20column purification. FIGS. 22 and 24 show the HPLC chromatograms at 280nm and 368 nm, respectively, of the purified non-polar proanthocyanidins(“fraction 8”). The peaks in FIG. 21 marked with asterisks arenon-proanthocyanidin flavonoid compounds based on their UV spectra.These compounds are reduced in the purified non-polar product (“fraction8”) isolated after Sephadex LH-20 column as shown HPLC chromatogram at280 nm in FIG. 22. The effect of the gel purification can be better seenby comparing the HPLC chromatogram at 368 nm of the non-polarproanthocyanidins before purification (FIG. 23). Thenon-proanthocyanidin impurities appear in FIGS. 23 at 4-6 minutes and15-17 minutes. Except for a small amount of the flavonoid compoundeluting at 5.8 minutes, there is no trace of flavonoid compounds in thepurified sample as shown in FIG. 24. A ¹³C NMR spectrum of the purifiednon-polar proanthocyanidin “fraction 8” is shown in FIG. 25. FIG. 33shows an IR spectrum of fraction 7, and FIG. 34 shows an IR spectrum of“fraction 8.”

Example 13 Purification of Blueberry Polar and Non-PolarProanthocyanidins by VLC Followed by Semi-Preparative HPLC

[0174] The starting material for this example was a totalphenol-enriched “fraction 3” prepared from blueberries and isolatedduring the 70% ethanol elution from a brominated polystyrene resin. Aportion (6.00 g) of “fraction 3” was dissolved in 80 mL of water andloaded onto a 30 mL C-18 VLC column as described previously. The loadingeluent was collected and combined with 100 mL of a 0.1% TFA wash eluent(“fraction 5”). Next, the column was washed with 80 mL of 40% methanolto remove residual polar compounds (“fraction 5”) and then with 80 mL of70% methanol to give the non-polar proanthocyanidin fraction (“fraction6”). Table 7 summarizes the results of this experiment. TABLE 7Purification of blueberry proanthocyanidins % Proantho-Proanthocyanidins cyanidins Sample Solids (g) (mg) purity “fraction 3”6.00 1614 27 Loading Eluent + Wash 2.11 899 43 40% MeOH fraction 2.46580 24 70% MeOH fraction 0.67 323 48

[0175] The polar proanthocyanidins “fraction 5” (loading eluent+wash)and the non-polar proanthocyanidins fraction 6 (70% methanol elution)were each further purified by semi-preparative HPLC by the methoddescribed in Example 12 to provide “fraction 7” and “fraction 8”,respectively. The HPLC chromatograms at 280 nm of the blueberry polarproanthocyanidins fraction before and after the semi-preparativepurification (i.e., “fraction 5” and “fraction 7”) are shown in FIGS. 26and 27, respectively. The HPLC chromatograms at 280 nm of the blueberrynon-polar proanthocyanidins fraction before and after thesemi-preparative purification (i.e., “fraction 6” and “fraction 8”) areshown in FIGS. 28 and 29, respectively. The semi-preparativepurifications of both the polar and non-polar fractions removedundesired anthocyanins and polar flavonoid compounds from theproanthocyanidins, as evidenced by the absence of peaks between about 0and 8 minutes in FIGS. 27 and 29. FIG. 37 shows an IR spectrum of“fraction 7,” and FIG. 38 shows an IR spectrum of “fraction 8.”

Example 14 Purification of Plum Polar and Non-Polar Proanthocyanidins byVLC Followed by Semi-Preparative HPLC

[0176] The starting material for this example was a combination of“fraction 3” and “fraction 4” isolated from plums and containingapproximately 17% total proanthocyanidins, of which 61% were designatedas polar and 39% as non-polar. A portion (8.00 g) of this compositionwas dissolved in 100 mL of water containing 0.5% TFA and loaded onto a45 mL C-18 VLC column as described previously. The loading eluent wascollected, and the column was washed with 50 mL of 0.1% TFA. The loadingeluent and wash fractions were combined to provide the polarproanthocyanidins fraction (“fraction 5”). An HPLC of the polarproanthocyanidin “fraction 5” is shown in FIG. 30. The column was elutedwith 100 mL of 40% methanol containing 0.5% TFA followed by 100 mL of70% methanol containing 0.5% TFA. All methanol fractions were combinedto provide the non-polar proanthocyanidin fraction (“fraction 6”). Table8 summarizes the results of this experiment. TABLE 8 Purification ofplum proanthocyanidins % Proantho- Proanthocyanidins cyanidins SampleSolids (g) (mg) purity Plum fractions 3 and 4 8.00 1328 17 LoadingEluent + Wash 4.32 651 15 40% MeOH fraction 3.76 486 13 70% MeOHfraction 0.45 300 67

[0177] The polar proanthocyanidin “fraction 5” (combined loading eluentand wash eluent) was further purified by semi-preparative HPLC by themethod described in Example 12 to provide “fraction 7.” Removal ofanthocyanins and other more polar impurities increased theproanthocyanidin purity of the sample from 15% to 100%. The HPLCchromatogram at 280 nm of the purified polar proanthocyanidin “fraction7” is shown in FIG. 31. The non-polar “fraction 6” (combined 40% and 70%methanol washes) was not purified further. The HPLC chromatogram at 280nm of the non-polar proanthocyanidin “fraction 6” is shown in FIG. 32.FIG. 39 is an IR spectrum of “fraction 7” and FIG. 40 is an IR spectrumof “fraction 6”.

Example 15 Purification of Proanthocyanidins Fraction from ElderberryVLC Fraction

[0178] A VLC column was prepared using Amberchrom CG-71cd resin (80-160μm particle size, TosoHaas; Philadelphia, Pa.). A water extract ofelderberry was prepared and a portion of this extract was loaded ontothe VLC column. The column was then washed with water and eluted using30%, 40%, 50%, 60%, 70%, and 100% methanol. All fractions eluted withmethanol were retained separately. The VLC fraction eluted with 50%methanol was evaporated on a rotary evaporator to remove the methanoland then lyophilized to remove the water. The dried material was groundto a powder using a mortar and pestle. The dried sample was assayed byHPLC using the method as described in Example 10. Using the results ofthis assay, a semi-preparative HPLC method was derived from theanalytical HPLC method to isolate the proanthocyanidins. The mobilephase was: channel A=100% acetonitrile; channel B=0.5% trifluoroaceticacid in water; channel C=100% methanol. The flow rate was set at 30mL/min. The gradient employed is provided in Table 9. TABLE 9 HPLCgradient for purification of elderberry proanthocyanidins Time (min) % A% B % C 0.0 11.0 81.0 8.0 9.0 11.0 81.0 8.0 17.0 34.0 58.0 8.0 22.0 34.058.0 8.0 23.0 92.0 0.0 8.0 28.0 92.0 0.0 8.0 28.1 11.0 81.0 8.0 36.011.0 81.0 8.0

[0179] Approximately 500 mg of the dried material was dissolved in waterat a solids concentration of approximately 50 mg/mL. A very smallinjection was made to determine the retention time of the relevantpeaks. Based on this initial injection, two peaks were collected: PeakA, which eluted between 14 and 22 minutes and Peak B, which elutedbetween 26 and 28 minutes. Five injections of the concentrated solutionwere made, and the appropriate collections of each peak were pooled fromeach injection. The sample obtained by the collection of Peak A wasdetermined to contain the proanthocyanidins and was evaporated to removethe organic solvents and a portion of the water. The concentrated samplewas assayed using the HPLC method as described in Example 10. Thechromatographic purity of the sample was determined to be 93.9%. Thesample was then lyophilized to obtain the dry material. Once dry, asmall portion of the sample was brought up in 70% ethanol at aconcentration of 1.918 mg/mL and re-assayed by the same HPLC method.Using the results of this analysis and the previously obtainedchromatographic purity, a peak area response factor was determined. Thisinformation was used to determine the proanthocyanidins concentration inother purified fractions. The HPLC chromatogram at 280 nm of theproanthocyanidin “standard” is shown in FIG. 14.

Example 16 Purification of Cranberry Proanthocyanidins by VLC Followedby Semi-Preparative HPLC

[0180] The starting material for this example was 8.00 g of purifiedcranberry extract (“fraction 3”+“fraction 4”) comprising 14% totalproanthocyanidins. This material was dissolved in 100 mL of watercontaining 1 mL of trifluoroacetic acid and loaded onto a 50 mL C-18 VLCcolumn as described previously. The loading eluent (100 mL) wascollected and combined with 50 mL of 0.1% TFA wash eluent to obtain“fraction 5”. Next the column was washed with 100 mL of 40% methanol toremove residual polar compounds and eluted with 100 mL of 70% methanolto give the non-polar proanthocyanidins “fraction 6”. Table 10summarizes the results of this experiment. TABLE 10 Purification ofcranberry proanthocyanidins % Proantho- Proanthocyanidins cyanidinsSample Solids (g) (mg) purity Cranberry fractions 3 + 4 8.00 1514 14.3Loading Eluent + Wash 3.60 748 20.8 40% MeOH fraction 3.39 677 20.0 70%MeOH fraction 0.44 93 21.1

[0181] The polar proanthocyanidins fraction (loading eluent+wash) wasfurther purified by semi-preparative HPLC by the method described inExample 12 to obtain “fraction 7”. FIG. 41 is an HPLC chromatogram ofthe polar proanthocyanidins fraction before semi-preparativepurification, and FIG. 42 is an HPLC chromatogram of the polarproanthocyanidins fraction after purification. FIG. 43 is an HPLCchromatogram of the non-polar proanthocyanidins fraction. Polarnon-proanthocyanidin compounds such as anthocyanins that eluted beforethe proanthocyanidins were removed in this process.

Example 17 Herpes Simplex Virus 2 Assay of Elderberry Fractions

[0182] The antiviral activities of a crude elderberry extract andfractions 1, 3 and 4 isolated as described in Example 6 were determinedusing the viral cytopathic effect (CPE) assay. This assay has previouslybeen described (Wyde, et al., Drug Develp. Res. 28:467-472 (1993)). Allantiviral activities are reported as 50% effective dose (ED₅₀).

[0183] Table 11 summarizes the ED₅₀ for CPE inhibition for the fourcompositions tested. TABLE 11 ED₅₀ for CPE inhibition of elderberryfractions CPE Inhibition Composition (ED₅₀) Crude extract >100 μg/mL“fraction 1” >100 μg/mL “fraction 3” >100 μg/mL “fraction 4” <0.03μg/mL 

Example 18 Viral Assays

[0184] Total phenol-enriched compositions of this invention preparedfrom fruits and berries have demonstrated broad activity against avariety of DNA and RNA viruses and are suitable as active ingredientsuseful in treating inflammation in humans and animals. In cell culture,the enriched compositions exhibit potent activity against isolates andlaboratory strains of respiratory syncytial virus (RSV), influenza A andB virus, parainfluenza virus (PIV), as well as other respiratory andHerpes simplex viruses. Total phenol-enriched compositions are suitableas active ingredients useful in treating a wide range of viralinfections in humans and animals.

[0185] Assays used to measure activity against each virus are well knownto those skilled in the art. Minced specific target tissue was exposedto the desired virus and the rate of growth of the virus was measured inthe presence and in the absence of the test materials. The antiviralactivities of purified proanthocyanidin-enriched compositions preparedfrom various fruits and berries were determined.

[0186] Cell lines: The viral assays used the following cells/cell linesin determining relative ED₅₀ (50% effective dose) or 50% inhibitoryendpoints: RSV (respiratory syncytial virus) and PIV (parainfluenzavirus) assays used MA-104 cells originating from African green monkeykidneys; Influenza A and B assays used MDCK cells originating fromcanine kidneys; Rhinovirus assays used HeLa and KB cells; Herpes simplexviruses 1 and 2 used HHF cells taken from human foreskin fibroblasts;West Nile viral assays used Vero cells taken from African green monkeykidneys; Adenovirus type 1 assays used A549 cells originating from humanlung carcinoma; and Punta Toro A assays used LLC-MK2 cells originatingfrom Rhesus monkey kidneys.

[0187] The assays used known drug standards (ribivarin or acyclovir) aspositive controls. The ED₅₀s for ribivarin in the assays used in thisExample are as follows: RSV (respiratory syncytial virus) assay ED₅₀=20μg/mL; PVI (parainfluenza virus) assay ED₅₀=20 μg/mL; Influenza A and Bassays ED₅₀=2-3 μg/mL; Rhinovirus assay ED₅₀<μg/mL; West Nile viralassay ED₅₀=20 μg/mL; Adenovirus type 1 assay ED₅₀=10 μg/mL; and PuntaToro A assay ED₅₀=20 μg/mL. Herpes simplex 1 and 2 assays used acycloviras a positive control, which has an ED₅₀ of 1-2 μg/mL in the HSV1 andHSV2 assays.

[0188] The data obtained in the viral assays for certain compositions ofthis invention are provided in Table 12. In cell cultures, thecompositions exhibited potent activity against isolates and laboratorystrains of influenza A virus (strains H1N1 and H3N3), influenza B virus,adenovirus type 1, Punta Toro A virus, and Rhinovirus type 2. Comparisonof the bioactivity data in Table 12 to acyclovir and ribavirin in theantiviral screenings clearly shows that the compositions of thisinvention are biologically active in these assays and compete favorablywith the well-established pharmaceuticals used to treat these viraldiseases. TABLE 12 ED₅₀'s (μg/mL) of various fractions in a COX-2 assayVirus Influenza Influenza A A Influenza Adenovirus Punta Rhinovirus WestNile Varicella- Source Fraction (H1N1) (H3N2) B Type 1 Toro A Type 2Virus zoster virus HSV-1 HSV-2 Cranberry 4 3.2 3.2 3.2 20 5.6 61 15 Plum4 32 32 32 25 70 70 32 Blueberry 4 32 32 32 30 25 30 31 45 11.4Elderberry 4 28-55 28-55 88 45 11 Elderberry 6 15 Elderberry 7 inactiveinactive inactive Elderberry 8 28 35 Elderberry Fig 14 inactive 0.0867.7 49 Grape* NA 4 6 3.2 20 6.8

Example 19 Evaluation of COX-2 Activity of Total Phenol-EnrichedCompositions

[0189] Cyclooxygenase enzymes (COX-1 and COX-2) catalyze the conversionof arachidonic acid and other essential fatty acids into variousprostaglandins. Prostaglandins are hormone-like substances responsiblefor inflammation in mammals. Inhibition of the COX-2 enzymes can reduceinflammation in tissue with minimal side effects. On the other hand,inhibition of COX-1 causes gastric ulceration and other undesirable sideeffects in the body. Complete inhibition of the COX-1 enzyme is notdesirable. Compounds that selectively inhibit COX-2 enzyme are betteranti-inflammatory agents. Total phenol-enriched compositions of thisinvention prepared from fruits and berries have been shown to inhibitthe COX-2 enzyme, and are suitable as active ingredients useful intreating inflammation in humans and animals.

[0190] In this assay the material to be assayed was mixed with mincedspecific murine or bovine organ tissues that are known to contain thedesired enzyme. Arachidonic acid was added to this mixture. The rate ofuptake of oxygen is measured and compared with the rate of uptakeobserved with known COX inhibitors. The COX-2 assay is based onquantitative production of PGE₂ from arachidonic acid using humanrecombinant COX-2 positive cells.

[0191] The results for several compositions are shown in Table 13.Comparison of the data for the compositions shown in Table 13 withdetermined COX-2 bioactivities of known drug standards (aspirin andindomethacin) clearly shows that the purified proanthocyanidin-enrichedcompositions of this invention are biologically active in the COX-2assay. Aspirin is active against COX-2 at 660 μg/mL and against COX-1 at240 μg/mL. Indomethacin is active against COX-2 at 10 μg/mL. Thecompositions in Table 13 therefore show a 2.5 to 6 fold increase inpotency in the COX-2 assay over the most commonly used treatment forinflammation (i.e., aspirin), and are indicative of the utility of thepurified proanthocyanidin-enriched compositions of this invention intreating inflammation in mammals. TABLE 13 COX-2 activities forproanthocyanidin enriched compositions Source Fraction IC₅₀ (μg/mL)Blueberry 3 108 Cranberry 4 218 Grape* N/A 275 Elderberry 4 >1000 Plum 4>1000

We claim:
 1. A method of preparing a composition enriched in totalphenols, comprising: a) providing a crude extract of one or more plantmaterials that contain phenolic compounds, said extract comprisingproanthocyanidins, anthocyanins, and non-phenolic compounds; b)filtering said crude extract; c) contacting said filtered extract with abrominated polystyrene resin, wherein said resin releasably adsorbs saidphenols but does not retain said non-phenolic compounds; d) washing saidresin with a wash eluent to elute said non-phenolic compounds; e)eluting the resin with a first eluent and collecting a first fractioncontaining phenols; f) eluting the resin with a second eluent andcollecting a second fraction containing phenols; and g) isolating thefraction from step e) or step f) or combining said fractions from stepse) and f) to obtain a composition enriched in total phenols, whereinsaid composition has substantially depleted levels of said non-phenoliccompounds.
 2. The method of claim 1, wherein said crude extract isprepared by extracting dried or fresh plant material(s) with anacidified extraction solvent.
 3. The method of claim 2, wherein saidacidified extraction solvent comprises an aqueous solution havingbetween about 0-95% ethanol and between about 0-3% acid.
 4. The methodof claim 3, wherein said acid is sulfuric acid, acetic acid orhydrochloric acid.
 5. The method of claim 2, wherein said acidifiedextraction solvent comprises an aqueous solution having between about0-100% methanol and between about 0-3% acid.
 6. The method of claim 5,wherein said acid is sulfuric acid, acetic acid or hydrochloric acid. 7.The method of claim 1, wherein said wash eluent contains at least 0.003%acid.
 8. The method of claim 7, wherein said acid is acetic acid,hydrochloric acid or sulfuric acid.
 9. The method of claim 1, whereinsaid first eluent comprises between about 50 and 70% ethanol in waterand 0.003% acid.
 10. The method of claim 9, wherein said acid is aceticacid, hydrochloric acid or sulfuric acid.
 11. The method of claim 1,Wherein said second eluent comprises between about 70 and 90% ethanol inwater.
 12. The method of claim 1, wherein said composition comprisesbetween about 10-80% total phenols.
 13. The method of claim 12, whereinsaid composition comprises at least 12% total phenols.
 14. The method ofclaim 12, wherein said composition comprises at least 25% total phenols.15. The method of claim 1, wherein said composition produces peaks inthe region between 60 and 75 minutes in HPLC traces substantially asillustrated in FIGS. 12 and
 13. 16. The method of claim 1, wherein saidplant material is fruits and/or berries.
 17. The method of claim 16,wherein said fruits and/or berries are selected from the groupconsisting of elderberries, blueberries, bilberries, blackberries,strawberries, cranberries, raspberries, plums, red currants, blackcurrants, cherries, and grapes.
 18. The method of claim 1, wherein step(a) further comprises adding pectinase to said crude extract.
 19. Themethod of claim 18, wherein said pectinase is present in an amountbetween about 0 and 0.12% by weight of said plant material.
 20. Themethod of claim 1, further comprising adding an excipient to saidcomposition.
 21. The method of claim 20, wherein said excipient isselected from the group consisting of preservatives, carriers, bufferingagents, thickening agents, suspending agents, stabilizing agents,wetting agents, emulsifying agents, coloring agents and flavoringagents.
 22. The method of claim 1, further comprising: h) loading saidcomposition from step e), said composition from step f), or saidcomposition from step g) onto a low pressure vacuum liquidchromatography column packed with a reversed-phase lipophilic resin andcollecting fractions that elute during said loading; i) eluting saidresin with water; j) combining fractions from steps h) and i) to obtaina first composition enriched in polar proanthocyanidins; and k) elutingsaid resin with increasing amounts of a polar organic solvent to obtaina second composition enriched in non-polar proanthocyanidins.
 23. Themethod of claim 22, further comprising purifying said first compositionby reversed-phase preparative HPLC to obtain said more polarproanthocyanidins substantially free of anthocyanins.
 24. The method ofclaim 33, further comprising purifying said second composition by gelfiltration or preparative HPLC to obtain said less polarproanthocyanidins substantially free of anthocyanins.
 25. A compositionenriched in total phenols prepared by the method of claim
 1. 26. Acomposition enriched in polar proanthocyanidins prepared by the methodof claim
 22. 27. The composition of claim 25, wherein said plantmaterial is fruits and/or berries.
 28. The composition of claim 26,wherein said fruits and/or berries are selected from the groupcomprising elderberries, blueberries, bilberries, blackberries,strawberries, cranberries, raspberries, plums, red currants, blackcurrants, cherries, and grapes..
 29. The composition of claim 27,wherein said composition has a total phenol concentration greater than12.5%.
 30. A composition enriched in polar proanthocyanidins prepared bythe method of claim
 23. 31. A composition enriched in non-polarproanthocyanidins prepared by the method of claim
 22. 32. A compositionenriched in non-polar proanthocyanidins prepared by the method of claim24.
 33. A composition enriched in total phenols, characterized by anHPLC chromatogram having peaks in the region between 60 and 75 minutessubstantially as illustrated in FIGS. 12 and
 13. 34. The composition ofclaim 33, further characterized by the capability of exerting anantiviral effect as demonstrated in in vitro assays for antiviralactivity against a virus selected from the group consisting of influenzavirus type A, influenza virus type B, rhinovirus type 2, Herpes simplexvirus 1, Herpes simplex virus 2, parainfluenza virus, West Nile virus,Varicella-zoster virus, Rhinovirus Type 2, Adenovirus Type I, and PuntaToro A virus.
 35. The composition of claim 33, further characterized bythe capability of exerting an anti-inflammatory effect as demonstratedin in vitro assays for anti-inflammatory activity.
 36. The compositionof claim 33, further characterized by the capability of exerting anantimicrobial effect as demonstrated in in vitro assays forantimicrobial activity.
 37. The composition of claim 33, furthercomprising one or more immunoactive agents.
 38. The composition of claim37, wherein said immunoactive agent is selected from the groupconsisting of arabinogalactan, species of Echinacea, vitamins, mineralspolysaccharides and astragalus.
 39. The composition of claim 33, furthercomprising one or more antimutagenic agents.
 40. The composition ofclaim 39, wherein said antimutagenic agent is selected from the groupconsisting of green tea extracts, catechins, epicatechins,epigallocatechins, gallocatechins, and flavonoids.
 41. A method fortreating symptoms in a mammal caused by an infecting organism or agentcomprising administering an effective amount of a composition isolatedaccording to the method of claim
 1. 42. The method of claim 41, whereinsaid organism is a virus selected from the group consisting of influenzavirus type A, influenza virus type B, rhinovirus type 2, Herpes simplexvirus 1, Herpes simplex virus 2, parainfluenza virus, West Nile virus,Varicella-zoster virus, Rhinovirus Type 2, Adenovirus Type I, and PuntaToro A virus.
 43. A method for treating symptoms in a mammal caused by anonviral microbial infection comprising administering an effectiveamount of a composition isolated according to the method of claim 1.